Compounds for altering mitochondrial function and cellular responses

ABSTRACT

Compounds for treating diseases by altering mitochondrial function that affects cellular processes, as well as to compositions and methods related thereto. The compounds have the structure  
                 
 
     wherein R 1 , R 2 , R 3  and A are as defined herein.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention generally relates to compounds for treatingdiseases by altering mitochondrial function that affects cellularprocesses, as well as to compositions and methods related thereto.

[0003] 2. Description of the Related Art

[0004] Mitochondria are the subcellular organelles that are the mainenergy source in cells of higher organisms, and provide direct andindirect biochemical regulation of a wide array of cellular respiratory,oxidative and metabolic processes. The electron transport chain (ETC)machinery resides in the mitochondrion, and drives oxidativephosphorylation to produce metabolic energy in the form of adenosinetriphosphate (ATP). Mitochondria also play a critical role inmaintaining intracellular calcium homeostasis. In addition to their rolein energy production in growing cells, mitochondria (or, at least,mitochondrial components) are required for at least some forms ofprogrammed cell death (PCD), also known as apoptosis (Newmeyer et al.,Cell 79:353-364, 1994; Liu et al., Cell 86:147-157, 1996). Apoptosis isrequired for normal development of the nervous system and functioning ofthe immune system. However, some disease states are thought to beassociated with either insufficient or excessive levels of apoptosis(e.g., cancer and autoimmune diseases in the first instance, and strokedamage and neurodegeneration in Alzheimer's disease in the latter case).For general reviews of apoptosis, and the role of mitochondria therein,see Green and Reed (Science 281:1309-1312, 1998), Green (Cell94:695-698, 1998) and Kroemer (Nature Medicine 3:614-620, 1997).

[0005] Defective mitochondrial activity, including but not limited tofailure at any step of the elaborate multi-complex mitochondrialassembly, known as the electron transport chain (ETC), may result in (i)decreases in ATP production, (ii) increases in the generation of highlyreactive free radicals (e.g., superoxide, peroxynitrite and hydroxylradicals, and hydrogen peroxide), (iii) disturbances in intracellularcalcium homeostasis and (iv) the release of factors that initiate theapoptosis cascade. Because of these biochemical changes, mitochondrialdysfunction has the potential to cause widespread damage to cells andtissues. For example, oxygen free radical induced lipid peroxidation isa well established pathogenic mechanism in central nervous system (CNS)injury such as that found in a number of degenerative diseases, and inischemia (i.e., stroke).

[0006] Cells from long-lived tissue that have high energy demands suchas neurons, pancreatic islet cells, cardiac and muscle cells areparticularly vulnerable to mitochondrial dysfunction. A number ofdegenerative diseases may thus be caused by or associated with eitherdirect or indirect alterations in mitochondrial function. These includeAlzheimer's Disease, diabetes mellitus, Parkinson's Disease, neuronaland cardiac ischemia, Huntington's disease and other relatedpolyglutamine diseases (spinalbulbar muscular atrophy, Machado-Josephdisease (SCA-3), dentatorubro-pallidoluysian atrophy (DRPLA) andspinocerebellar ataxias, dystonia, Leber's hereditary optic neuropathy,schizophrenia, and myodegenerative disorders such as mitochondrialencephalopathy, lactic acidosis, and stroke (MELAS), and myoclonicepilepsy ragged red fiber syndrome (MERRF).

[0007] Increasing evidence points to the fundamental role ofmitochondrial dysfunction in neurodegenerative diseases (Beal, Biochim.Biophys. Acta 1366: 211-223, 1998), and recent studies implicatemitochondria for regulating the events that lead to necrotic andapoptotic cell death (Susin et al., Biochim. Biophys. Acta 1366:151-168, 1998). Stressed (stressors include free radicals, highintracellular calcium, loss of ATP, among others) mitochondria mayrelease pre-formed soluble factors that can initiate apoptosis throughan interaction with novel apoptosomes (Marchetti et al., Cancer Res.56:2033-38, 1996; Li et al., Cell 91: 479-89, 1997). Release ofpreformed soluble factors by stressed mitochondria, like cytochrome c,may occur as a consequence of a number events. In some cases, release ofapoptotic molecules (apoptogens) occurs when mitochondria undergo asudden change in permeability to cytosolic solutes. This process hasbeen termed “permeability transition”. There is strong evidence thatsuggests that the loss of mitochondrial function may be due to theactivation of the mitochondrial permeability transition pore, a Ca²⁺regulated inner membrane megachannel. Opening of the mitochondrialpermeability transition pore results in the exchange of solutes that areless than 1500 daltons in size, collapse of the mitochondrial membranepotential, and uncoupling of the electron transport chain. In othercases, the permeability may be more subtle and perhaps more localized torestricted regions of a mitochondrion. In still other cases, overtpermeability transition may not occur but apoptogens can still bereleased as a consequence of mitochondrial abnormalities. The magnitudeof stress (ROS, intracellular calcium levels) influences the changes inmitochondrial physiology that ultimately determine whether cell deathoccurs via a necrotic or apoptotic pathway. To the extent that apoptoticcell death is a prominent feature of degenerative diseases,mitochondrial dysfunction may be a critical factor in diseaseprogression.

[0008] Whereas mitochondria-mediated apoptosis may be critical indegenerative diseases, it is thought that disorders such as cancerinvolve the unregulated and undesirable growth (hyperproliferation) ofcells that have somehow escaped a mechanism that normally triggersapoptosis in such undesirable cells. Enhanced expression of theanti-apoptotic protein, Bcl-2 and its homologues is involved in thepathogenesis of numerous human cancers. Bcl-2 acts by inhibitingprogrammed cell death and overexpression of Bcl-2 and the related Bcl-xLblock mitochondrial release of cytochrome c from mitochondria and theactivation of caspase 3 (Yang et al, Science 275:1129-1132, 1997; Klucket al., Science 275:1132-1136, 1997; Kharbanda et al., Proc. Natl. Acad.Sci. USA 94:6939-6942, 1997). Bcl-2 also binds to several proteins thatare involved in death regulation (Reed, Nature 387:773-779, 1997). Overexpression of Bcl-2 and Bcl-xL protect against the mitochondrialdysfunction preceding nuclear apoptosis that is induced bychemotherapeutic agents. In addition, acquired multi-drug resistance tocytotoxic drugs is associated with inhibition cytochrome c release thatis dependent on overexpression of Bcl-xL (Kojima et al., J. Biol. Chem.273: 16647-16650, 1998). Because mitochondria have been implicated inapoptosis, it is expected that agents that interact with mitochondrialcomponents will effect a cell's capacity to undergo apoptosis. Thus,agents that induce or promote apoptosis in hyperproliferative cells areexpected to be useful in treating such hyperproliferative disorders anddiseases.

[0009] Thus, alteration of mitochondrial function has great potentialfor a broad-based therapeutic strategy for designing drugs to treatdegenerative diseases as well as hyperproliferative diseases. Themitochondrial permeability transition pore is a key target for theprevention of mitochondrial function collapse that leads to cell death,or the induction of apoptosis in cancer cells via dysregulation ofmitochondria. This megachannel is a multi-protein complex in which theadenine nucleotide translocator (ANT) has been implicated as thecritical molecular component.

[0010] ANT is located in the inner mitochondrial inner membrane and itfacilitates transport of ADP and ATP across the mitochondrial innermembrane. In humans there are three genetic isoforms (ANT1, ANT2 andANT3), each with different tissue expression patterns. ANTI is highlyexpressed in heart and skeletal muscle and is induced during myoblastdifferentiation. ANT2 is overexpressed in a variety ofhyperproliferative cells, tumors and neoplastically transformed cellswith high glycolytic rates (Battini et al., J. Biol. Chem.262:4355-4359, 1987; Torroni et al., J. Biol. Chem. 265:20589-20593,1990; Faure-Vigny et al., Mol. Carcinogen. 16:165-172, 1996; Heddi etal., Biochim. Biophys. Acta 1316:203-209, 1996; and Giraud et al., J.Mol. Biol. 281:409-418, 1998). ANT3 is ubiquitously expressed in alltissues. ANT3 transcript level is proportional to the level of oxidativemetabolism in a given tissue.

[0011] Two different conformations of ANT have been demonstrated on thebasis of interactions with specific ligands, namely the inhibitorscarboxyatractyloside (CATR) and bongkrekic acid (BKA). Ligands can bindto ANT in an asymmetric fashion, either from the matrix (m) or from thecytosolic (c) side of the inner mitochondrial membrane. For example,CATR binds to ANT in the c-conformation and induces permeabilitytransition, while BKA interacts with ANT in the m-conformation andinhibits permeability transition in response to a variety of apoptoticstimuli (see, Budd et al., PNAS US 97:6161-6166, 2000) Different smallmolecule ligands of ANT isoforms therefore can possess a spectrum ofactivities—that is, they can act as cell protective agents targeted fordegenerative diseases and as cytotoxic agents for hyperproliferativediseases.

[0012] Bongkrekic acid (BKA) is a polyenoic triacid that is produced bythe microorganism Pseudomonas cocovenenans.

[0013] In the protonated form, BKA diffuses through the lipid phase ofthe inner mitochondrial membrane and binds to the matrix side of ANTwith high affinity (2×10⁻⁸ M). Upon binding, BKA is believed tostabilize ANT in the m-conformation. Accordingly, BKA is believed tohave significant potential as a cell-protective agent. Unfortunately,there are a number of problems associated with BKA. For example, largequantities of BKA are difficult to obtain by fermentation. While aconvergent total synthesis of BKA has been reported (J. Am. Chem. Soc.106:462-463, 1984), this technique involves 33 steps which makes ittedious to produce.

[0014] Accordingly, there is a need for agents that are cytotoxic withregard to undesirable cells and tissues, as well as agents that limit orprevent damage to desirable organelles, cells and tissues resulting fromvarious consequences of mitochondrial dysfunction. In the formerinstance, such agents are desired to treat hyperproliferative diseasesand disorders, or as species-specific antibiotics, herbicides orinsecticides. In the latter instance, because mitochondria are essentialorganelles for producing metabolic energy, agents that protectmitochondria against injury are desired for the prevention, treatmentand management of degenerative diseases, including mitochondriaassociated diseases. The present invention fulfills these needs andprovides other related advantages.

BRIEF SUMMARY OF THE INVENTION

[0015] In brief, the present invention generally relates to compoundswhich inhibit mitochondrial permeability transition by binding to theadenine nucleotide translocator (ANT) in the m-conformation, and therebyhave activity as cell-protective agents. In addition, this invention isdirected to compositions containing a compound of this invention incombination with a pharmaceutically acceptable carrier or diluent, aswell as to methods related to the administration of such compoundsand/or composition to an animal in need thereof (including humans).

[0016] The compounds of this invention have the following generalstructure (I):

[0017] including stereoisomers, prodrugs and pharmaceutically acceptablesalts thereof, wherein R₁, R₂, R₃ and A are as defined below.

[0018] The compounds of the invention are, in some aspects, mimics ofbongkrekic acid (BKA) and function as agonists or antagonists of proteintargets of BKA that elicit cell-protective or cytotoxic responses. Inone embodiment, the target(s) of BKA that is (are) affected by acompound of the invention is (are) one or more isoforms of ANT. In arelated embodiment, a compound of the invention binds preferentially toa specific isoform of ANT, but not to other ANT isoforms, from a singlespecies of organism.

[0019] In embodiments wherein a compound of the invention is cytotoxic,the compound has, for example, remedial, therapeutic, palliative,rehabilitative, preventative, disease-impeditive or prophylacticactivity with regard to hyperproliferative diseases and disorders suchas cancer, psoriasis and the like. In such embodiments, a cytotoxiccompound of the invention (a) binds preferentially to a specific isoformof ANT, but not to other ANT isoforms, from a single species oforganism, wherein the preferentially-bound isoform of ANT isoverexpressed in undesirable hyperproliferative cells; or (b)preferentially enters undesirable hyperproliferative cells.

[0020] In further embodiments wherein a compound of the invention iscytotoxic, the compound acts as a species-specific antibiotic, herbicideor insecticide. Such compounds binds preferentially to, respectively,(i) one or more ANT proteins from an undesirable parasitic or infectivespecies (e.g., a eukaryotic parasite such as members of the Trypanosomaor Leishmania genera), but not to the corresponding ANT protein(s) fromthe host species (i.e., a mammal such as a human); (ii) one or more ANTproteins from an undesirable plant species (e.g., a weed), but not tothe corresponding ANT protein(s) from desirable plants having economicvalue (e.g., crops or decorative plants), desirable insects (e.g., bees)or mammals including humans; or (iii) one or more ANT proteins from anundesirable insect (e.g., members of the genus Lepidoptera), but not tothe corresponding ANT protein(s) from desirable insects, desirableplants or mammals including humans.

[0021] In embodiments wherein a compound of the invention iscyto-protective, the compound is used, for example, to prevent, treat ormanage neurodegenerative diseases and disorders, (e.g., Alzheimer'sdisease and Parkinson's disease, or to ameliorate the undesirableeffects of acute events such as ischemia or cardiac arrest.

[0022] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings. To that end, various references are set forth hereinwhich describe in more detail certain aspects of this invention, and areeach incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

[0023] As noted above, the present invention is generally directed tocompounds and to pharmaceutical compositions containing the same, aswell as to methods for treating diseases by altering mitochondrialfunction that affects cellular processes. The compounds of thisinvention have the following general structure (I):

[0024] including stereoisomers, prodrugs and pharmaceutically acceptablesalts thereof,

[0025] wherein:

[0026] A is a direct bond, alkyldiyl, substituted alkyldiyl,—O-(alkyldiyl)-, —O-(substituted alkyldiyl)-, -(alkyldiyl)-O-,-(substituted alkyldiyl)-O-, —N(R′)-(alkyldiyl)-, —N(R′)-(substitutedalkyldiyl)-, -(alkyldiyl)-N(R′)-, -(substituted alkyldiyl)-N(R′)-,heterocyclediyl, substituted heterocyclediyl, heterocyclealkyldiyl orsubstituted heterocyclealkyldiyl, wherein R′ is hydrogen or alkyl;

[0027] R₁ is hydroxy, alkoxy, aryloxy, arylalkyloxy, amino, or mono- ordi-alkylamino;

[0028] R₂ is hydrogen, alkyl, substituted alky, aryl, substituted aryl,arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle,heterocylealkyl or substituted heterocyclealkyl; and

[0029] R₃ is alkyl, substituted alky, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocylealkyl or substituted heterocyclealkyl.

[0030] As used herein, the terms used above have the following meaning:

[0031] “Alkyl” means a straight chain or branched, saturated orunsaturated, cyclic or non-cyclic hydrocarbon having from 1 to 10 carbonatoms, while “lower alkyl” has the same meaning but only has from 1 to 6carbon atoms. Representative saturated straight chain alkyls includemethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; whilesaturated branched alkyls include isopropyl, sec-butyl, isobutyl,tert-butyl, isopentyl, and the like. Unsaturated alkyls contain at leastone double or triple bond between adjacent carbon atoms (also referredto as an “alkenyl” or “alkynyl”, respectively). Representative straightchain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; whilerepresentative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, (cycloalkyl)CH₂-, andthe like; while unsaturated cyclic alkyls include cyclopentenyl andcyclohexenyl, and the like. Cycloalkyls are also referred to herein as“carbocyclic” rings systems, and include bi- and tri-cyclic ring systemshaving from 8 to 14 carbon atoms such as a cycloalkyl (such as cyclopentane or cyclohexane) fused to one or more aromatic (such as phenyl)or non-aromatic (such as cyclohexane) carbocyclic rings.

[0032] “Alkyldiyl” means a divalent alkyl from which two hydrogen atomsare taken from the same carbon atom or from different carbon atoms,including divalent alkyl, alkenyl and alkynyl, as well as saturated andunsaturated carbocyclic rings as defined above.

[0033] “Halogen” means fluorine, chlorine, bromine or iodine.

[0034] “Oxo” means a carbonyl group (i.e., ═O).

[0035] “Mono- or di-alkylamino” means an amino substituted with onealkyl or with two alkyls, respectively.

[0036] “Alkoxy” means —O-(alkyl).

[0037] “Aryloxy” means —O-(aryl).

[0038] “Arylalkyloxy” means —O-(arylalkyl).

[0039] “Aryl” means an aromatic carbocyclic moiety such as phenyl ornaphthyl.

[0040] “Arylalkyl” means an alkyl having at least one alkyl hydrogenatom replaced with an aryl moiety, such as benzyl, —(CH₂)₂phenyl,—(CH₂)₃phenyl, —CH(phenyl)₂, and the like.

[0041] “Heteroaryl” means an aromatic heterocycle ring of 5- to 10members and having at least one heteroatom selected from nitrogen,oxygen and sulfur, and containing at least 1 carbon atom, including bothmono- and bicyclic ring systems. Representative heteroaryls are pyridyl,furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl,indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl,benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, andquinazolinyl.

[0042] “Heteroarylalkyl” means an alkyl having at least one alkylhydrogen atom replaced with a heteroaryl moiety, such as —CH₂pyridinyl,—CH₂pyrimidinyl, and the like. “Heterocycle” means a 5- to 7-memberedmonocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which iseither saturated, unsaturated, or aromatic, and which contains from 1 to4 heteroatoms independently selected from nitrogen, oxygen and sulfur,and wherein the nitrogen and sulfur heteroatoms may be optionallyoxidized, and the nitrogen heteroatom may be optionally quatemized,including bicyclic rings in which any of the above heterocycles arefused to a benzene ring. The heterocycle may be attached via anyheteroatom or carbon atom. Heterocycles include heteroaryls as definedabove. Thus, in addition to the heteroaryls listed above, heterocyclesalso include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

[0043] “Heterocyclediyl” means a divalent heterocycle from which twohydrogen atoms are taken from the same atom or from different atoms.

[0044] “Heterocyclealkyl” means an alkyl having at least one alkylhydrogen atom replaced with a heterocycle, such as —CH₂morpholinyl, andthe like.

[0045] “Heterocylcealkyldiyl” means a divalent heterocyclealkyl fromwhich two hydrogen atoms are taken from the same atom or from differentatoms.

[0046] The term “substituted” as used herein means any of the abovegroups (i.e., alkyl, aryl, arylalkyl, heterocycle, heterocyclealkyl,alkyldiyl, heterocyclediyl and heterocylcealkyldiyl) wherein at leastone hydrogen atom is replaced with a substituent. In the case of an oxosubstituent (“═O”) two hydrogen atoms are replaced. Substituents includehalogen, hydroxy, oxoalkyl, substituted alkyl (such as haloalkyl, mono-or di-substituted aminoalkyl, alkyloxyalkyl, and the like), aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,—NR_(a)R_(b), —NR_(a)C(═O)R_(b), —NR_(c)C(═O)NR_(a)R_(b),—NR_(a)C(═O)OR_(b) —NR_(a)SO₂R_(b), —OR_(a), —C(═O)R_(a) —C(═O)OR_(a)—C(═O)NR_(a)R_(b), —OC(═O)R_(a), —OC(═O)OR_(a), —OC(═O)NR_(a)R_(b),—NR_(a)SO₂R_(b), or a radical of the formula —Y—Z—R_(a) where Y isalkanediyl, substituted alkanediyl, or a direct bond, Z is —O—, —S—,—S(═O)-, —S(═O)₂-, —N(R_(b))-, —C(═O)-, —C(═O)O-, —OC(═O)-,—N(R_(b))C(═O)-, —C(═O)N(R_(b))- or a direct bond, wherein R_(a), R_(b)and R_(c) are the same or different and independently hydrogen, amino,alkyl, substituted alkyl (including halogenated alkyl), aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocylealkyl or substitutedheterocyclealkyl, or wherein R_(a) and R_(b) taken together with thenitrogen atom to which they are attached form a heterocycle orsubstituted heterocycle.

[0047] In one embodiment, A is a direct bond and the compounds have thefollowing structure (II):

[0048] In another embodiments, and depending upon the choice of A, thecompounds have one of the following structures (III) through (IX),wherein each of alkyldiyl, heterocyclediyl and/or heterocylcealkyldiylmoieties may be unsubstituted or substituted with one or moresubstituents as defined above.

[0049] The compounds of the present invention may generally be utilizedas the free acid or base. Alternatively, the compounds of this inventionmay be used in the form of acid or based addition salts. Acid additionsalts of the free base amino compounds of the present invention may beprepared by methods well known in the art, and may be formed fromorganic and inorganic acids. Suitable organic acids include maleic,fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic, oxalic,propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic,cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, andbenzenesulfonic acids. Suitable inorganic acids include hydrochloric,hydrobromic, sulfuric, phosphoric, and nitric acids. Based additionsalts include the ammonium ion, other suitable cations. Thus, the term“pharmaceutically acceptable salt” of structure (I) is intended toencompass any and all acceptable salt forms. Suitable salts in thiscontext may be found in Remington's Pharmaceuitcal Sciences, 17th ed.,Mack Publishing Co., Easton, Pa., 1985, which is hereby incorporated byreference.

[0050] In addition, prodrugs are also included within the context ofthis invention. Prodrugs are any covalently bonded carriers that releasea compound of structure (I) in vivo when such prodrug is administered toa patient. Prodrugs are generally prepared by modifying functionalgroups in a way such that the modification is cleaved, either by routinemanipulation or in vivo, yielding the parent compound.

[0051] With regard to stereoisomers, the compounds of structure (I) mayhave chiral centers and may occur as racemates, racemic mixtures and asindividual enantiomers or diastereomers. All such isomeric forms areincluded within the present invention, including mixtures thereof.Furthermore, some of the crystalline forms of the compounds of structure(I) may exist as polymorphs, which are included in the presentinvention. In addition, some of the compounds of structure (I) may alsoform solvates with water or other organic solvents. Such solvates aresimilarly included within the scope of this invention.

[0052] The compounds of this invention are typically formulated inconjunction with a suitable pharmaceutical carrier or diluent, and suchcompositions may be in any form that allows for the composition to beadministered to a patient. For example, the composition may be in theform of a solid, liquid or gas (aerosol). Typical routes ofadministration include, without limitation, oral, topical, parenteral(e.g., sublingually or buccally), sublingual, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal, intracavernous,intrameatal, intraurethral injection or infusion techniques. Thepharmaceutical composition is formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa patient take the form of one or more dosage units, where for example,a tablet may be a single dosage unit, and a container of one or morecompounds of the invention in aerosol form may hold a plurality ofdosage units.

[0053] For oral administration, which is the route of administration inpreferred embodiments, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

[0054] The composition may be in the form of a liquid, e.g., an elixir,syrup, solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain, inaddition to one or more agents that impair MCA activity, one or more ofa sweetening agent, preservatives, dye/colorant and flavor enhancer. Ina composition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

[0055] A liquid pharmaceutical composition as used herein, whether inthe form of a solution, suspension or other like form, may include oneor more of the following adjuvants: sterile diluents such as water forinjection, saline solution, preferably physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

[0056] A liquid composition intended for either parenteral or oraladministration should contain an amount of an agent that impairs MCAactivity as provided herein such that a suitable dosage will beobtained. Typically, this amount is at least 0.01 wt % of the agent inthe composition. When intended for oral administration, this amount maybe varied to be between 0.1 and about 70% of the weight of thecomposition. Preferred oral compositions contain between about 4% andabout 50% of the agent(s) that alter mitochondrial function. Preferredcompositions and preparations are prepared so that a parenteral dosageunit contains between 0.01 to 1% by weight of active compound.

[0057] The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the agent that impairs MCA activity of from about 0.1to about 10% w/v (weight per unit volume).

[0058] The composition may be intended for rectal administration, in theform, e.g., of a suppository that will melt in the rectum and releasethe drug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol. In the methods of the invention, the agent(s) that altermitochondrial function identified as described herein may beadministered through use of insert(s), bead(s), timed-releaseformulation(s), patch(es) or fast-release formulation(s).

[0059] It will be evident to those of ordinary skill in the art that theoptimal dosage of the compound may depend on the weight and physicalcondition of the patient; on the severity and longevity of the physicalcondition being treated; on the particular form of the activeingredient, the manner of administration and the composition employed.The use of the minimum dosage that is sufficient to provide effectivetherapy is usually preferred. Patients may generally be monitored fortherapeutic or prophylactic effectiveness using assays suitable for thecondition being treated or prevented, which will be familiar to thosehaving ordinary skill in the art and which, as noted above, willtypically involve determination of whether circulating insulin and/orglucose concentrations fall within acceptable parameters according towell-known techniques. Suitable dose sizes will vary with the size,condition and metabolism of the patient, but will typically range fromabout 10 mL to about 500 mL for 10-60 kg individual. It is to beunderstood that according to certain embodiments the agent may bemembrane permeable, preferably permeable through the plasma membraneand/or through mitochondrial outer and/or inner membranes. According tocertain other embodiments, the use of a compound as disclosed herein ina chemotherapeutic composition can involve such an agent being bound toanother compound, for example, a monoclonal or polyclonal antibody, aprotein or a liposome, which assist the delivery of said agent.

[0060] Another embodiment of the invention involves the creation andidentification of compounds that increase the degree or enhance the rateof apoptosis in hyperproliferative cells present in diseases anddisorders such as cancer and psoriasis (note that, for the purposes ofthe disclosure, the term “hyperproliferative disease or disorder”specifically excludes pregnancy). Because oncogenic changes rendercertain tumors more susceptible to apoptosis (Evan and Littlewood,Science 281:1317,1998), such agents are expected to be useful fortreating such hyperproliferative diseases or disorders. In a relatedembodiment, a biological sample from a patient having or suspected ofhaving a hyperproliferative disease or disorder are evaluated for theirsusceptibility to such agents using the methods of the invention. Cybridcells are a preferred biological sample in this embodiment.

[0061] A further embodiment of the invention involves the creation andidentification of agents that alter mitochondrial function and/orselectively affect MPT in mitochondria and/or cell death in aspecies-specific manner. By “species-specific manner” it is meant thatsuch agents affect MPT or cell death in a first organism belonging toone species but not in a second organism belonging to another species.This embodiment of the invention is used in a variety of methods.

[0062] For example, this embodiment of the invention to identify agentsthat selectively induce MPT and/or apoptosis in biological samplescomprising cells or mitochondria derived from different species, e.g.,in trypanasomes (Ashkenazi and Dixit, Science 281:1305, 1998), and othereukaryotic pathogens and parasites, including but not limited toinsects, but which do not induce MPT and/or apoptosis in their mammalianhosts. Such agents are expected to be useful for the prophylactic ortherapeutic management of such pathogens and parasites.

[0063] As another example, this embodiment of the invention is used tocreate and identify agents that selectively induce MPT and/or apoptosisin biological samples comprising cells or mitochondria derived fromundesirable plants (e.g., weeds) but not in desirable plants (e.g.,crops), or in undesirable insects (in particular, members of the familyLepidoptera and other crop-damaging insects) but not in desirableinsects (e.g., bees) or desirable plants. Such agents are expected to beuseful for the management and control of such undesirable plants andinsects. Cultured insect cells, including for example, the Sf9 and Sf21cell lines derived from Spodoptera frugiperda, and the HIGH FIVE (cellline from Trichopolusia ni (these three cell lines are available fromInVitrogen, Carlsbad, Calif.) may be biological sample in certain suchembodiments of the invention.

[0064] The suitability of a compound for treatment of a subject having adisease associated with altered mitochondrial function may be determinedby various assay methods. Such compounds are active in one or more ofthe following assays for measuring mitochondrial permeabilitytransition, or in any other assay known in the art that directly orindirectly measures induction of MPT, MPT itself or any downstreamsequelae of MPT, or that may be useful for identifying mitochondrialpermeability pore components (i.e., molecules that regulate MPT).Accordingly, it is also an aspect of the invention to providecompositions and methods for treating a disease associated with alteredmitochondrial function by administering a composition that regulatesMPT. In embodiments of the invention, agents to be formulated into suchcompositions may be identified by the following assay methods.

[0065] A. Assay for Mitochondrial Permeability Transition (MPT) Using2-,4-Dimethylaminostyryl-N-Methylpyridinium (DASPMI)

[0066] According to this assay, one may determine the ability of acompound of the invention to inhibit the loss of mitochondrial membranepotential that accompanies mitochondrial dysfunction. As noted above,maintenance of a mitochondrial membrane potential (ΔΨm) may becompromised as a consequence of mitochondrial dysfunction. This loss ofmembrane potential, or mitochondrial permeability transition (MPT), canbe quantitatively measured using the mitochondria-selective fluorescentprobe 2-,4-dimethylaminostyryl-N-methylpyridinium (DASPMI).

[0067] Upon introduction into cell cultures, DASPMI accumulates inmitochondria in a manner that is dependent on, and proportional to,mitochondrial membrane potential. If mitochondrial function is disruptedin such a manner as to compromise membrane potential, the fluorescentindicator compound leaks out of the membrane bounded organelle with aconcomitant loss of detectable fluorescence. Fluorimetric measurement ofthe rate of decay of mitochondria associated DASPMI fluorescenceprovides a quantitative measure of loss of membrane potential, or MPT.Because mitochondrial dysfunction may be the result of multiple factorsthat directly or indirectly induce MPT as described above (e.g., ROS,calcium flux), agents that retard the rate of loss of DASPMIfluorescence may be effective agents for treating diseases associatedwith altered mitochondrial function, according to the methods of thisinvention.

[0068] B. Assay of Apoptosis in Cells Treated with MitochondriaProtecting Agents

[0069] As noted above, mitochondrial dysfunction may be an inductionsignal for cellular apoptosis. According to this assay, one maydetermine the ability of a compound agent to inhibit or delay the onsetof apoptosis. Mitochondrial dysfunction may be present in cells known orsuspected of being derived from a subject having a disease associatedwith altered mitochondrial function, or mitochondrial dysfunction may beinduced in normal cells by one or more of a variety of physiological andbiochemical stimuli, with which those having skill in the art will befamiliar.

[0070] In one aspect of the apoptosis assay, cells that are suspected ofundergoing apoptosis may be examined for morphological, permeability orother changes that are indicative of an apoptotic state. For example,apoptosis in many cell types may cause altered morphological appearancesuch as plasma membrane blebbing, cell shape change, loss of substrateadhesion properties or other morphological changes that can be readilydetected by those skilled in the art using light microscopy. As anotherexample, cells undergoing apoptosis may exhibit fragmentation anddisintegration of chromosomes, which may be apparent by microscopyand/or through the use of DNA specific or chromatin specific dyes thatare known in the art, including fluorescent dyes. Such cells may alsoexhibit altered plasma membrane permeability properties as may bereadily detected through the use of vital dyes (e.g., propidium iodide,trypan blue) or by the detection of lactate dehydrogenase leakage intothe extracellular milieu. These and other means for detecting apoptoticcells by morphologic criteria, altered plasma membrane permeability andrelated changes will be apparent to those familiar with the art.

[0071] In another aspect of an apoptosis assay, translocation of cellmembrane phosphatidylserine (PS) from the inner to the outer leaflet ofthe plasma membrane is detected by measuring outer leaflet binding bythe PS-specific protein annexin (Martin et al., J. Exp. Med. 182:1545,1995; Fadok et al., J. Immunol. 148:2207, 1992.) In another aspect ofthe apoptosis assay, induction of specific protease activity in a familyof apoptosis-activated proteases known as the caspases is measured, forexample, by determination of caspase-mediated cleavage of specificallyrecognized protein substrates. These substrates may include, forexample, poly-(ADP-ribose) polymerase (PARP) or other naturallyoccurring or synthetic peptides and proteins cleaved by caspases thatare known in the art (see, e.g., Ellerby et al., J. Neurosci. 17:6165,1997). The synthetic peptide Z-Tyr—Val—Ala—Asp—AFC, wherein “Z”indicates a benzoyl carbonyl moiety and AFC indicates7-amino-4-trifluoromethylcoumarin (Kluck et al., Science 275:1132, 1997;Nicholson et al., Nature 376:37, 1995), is one such substrate. Othersubstrates include nuclear proteins such as U1-70 kDa and DNA-PKcs(Rosen and Casciola-Rosen, J. Cell. Biochem. 64:50, 1997; Cohen,Biochem. J. 326:1, 1997).

[0072] As described above, the mitochondrial inner membrane may exhibithighly selective and regulated permeability for many small molecules,including certain cations, but is impermeable to large (>˜10 kDa)molecules (see, e.g., Quinn, 1976 The Molecular Biology of CellMembranes, University Park Press, Baltimore, Md.). Thus, in anotheraspect of the apoptosis assay, detection of the mitochondrial proteincytochrome c that has leaked out of mitochondria in apoptotic cells mayprovide an apoptosis indicator that can be readily determined (Liu etal., Cell 86:147, 1996). Such detection of cytochrome c may be performedspectrophotometrically, immunochemically or by other well establishedmethods for determining the presence of a specific protein.

[0073] Release of cytochrome c from cells challenged with apoptoticstimuli (e.g., ionomycin, a well-known calcium ionophore) can befollowed by a variety of immunological methods. Matrix-assisted laserdesorption ionization time-of-flight (MALDI-TOF) mass spectrometrycoupled with affinity capture is particularly suitable for such analysissince apo-cytochrome c and holo-cytochrome c can be distinguished on thebasis of their unique molecular weights. For example, theSurface-Enhanced Laser Desorption/Ionization (SELDITM) system(Ciphergen, Palo Alto, Calif.) may be utilized to follow the inhibitionby mitochondria protecting agents of cytochrome c release frommitochondria in ionomycin treated cells. In this approach, a cytochromec specific antibody immobilized on a solid support is used to capturereleased cytochrome c present in a soluble cell extract. The capturedprotein is then encased in a matrix of an energy absorption molecule(EAM) and is desorbed from the solid support surface using pulsed laserexcitation. The molecular mass of the protein is determined by its timeof flight to the detector of the SELDITM mass spectrometer.

[0074] The person of ordinary skill in the art will readily appreciatethat there may be other suitable techniques for quantifying apoptosis,and such techniques for purposes of determining the effects ofmitochondria protecting agents on the induction and kinetics ofapoptosis are within the scope of the assays disclosed here.

[0075] C. Assay of Electron Transport Chain (ETC) Activity in IsolatedMitochondria

[0076] As described above, mitochondria associated diseases may becharacterized by impaired mitochondrial respiratory activity that may bethe direct or indirect consequence of elevated levels of reactive freeradicals such as ROS, of elevated cytosolic free calcium concentrationsor other stimuli. Accordingly, a compound for use in the treatment of adisease associated with altered mitochondrial function may restore orprevent further deterioration of ETC activity in mitochondria ofindividuals having mitochondria associated diseases. Assay methods formonitoring the enzymatic activities of mitochondrial ETC Complexes I,II, III, IV and ATP synthetase, and for monitoring oxygen consumption bymitochondria, are well known in the art (see, e.g., Parker et al.,Neurology 44:1090-96, 1994; Miller et al., J. Neurochem. 67:1897, 1996).It is within the scope of the methods provided by this invention toidentify a suitable compound using such assays of mitochondrialfunction, given the relationship between mitochondrial membranepotential and ETC activity as described above. Further, mitochondrialfunction may be monitored by measuring the oxidation state ofmitochondrial cytochrome c at 540 nm. Also as described above, oxidativedamage that may arise in mitochondria associated diseases may includedamage to mitochondrial components such that the oxidation state ofcytochrome c, by itself or in concert with other parameters ofmitochondrial function including, but not limited to, mitochondrialoxygen consumption, may be an indicator of reactive free radical damageto mitochondrial components. Accordingly, the invention provides variousassays designed to test the inhibition of such oxidative damage bycompounds that may influence mitochondrial membrane permeability. Thevarious forms such assays may take will be appreciated by those familiarwith the art, and are not intended to be limited by the disclosuresherein, including in the Examples.

[0077] For example, Complex IV activity may be determined usingcommercially available cytochrome c that is fully reduced via exposureto excess ascorbate. Cytochrome c oxidation may then be monitoredspectrophotometrically at 540 nm using a stirred cuvette in which theambient oxygen above the buffer is replaced with argon. Oxygen reductionin the cuvette may be concurrently monitored using a micro oxygenelectrode with which those skilled in the art will be familiar, wheresuch an electrode may be inserted into the cuvette in a manner thatpreserves the argon atmosphere of the sample, for example through asealed rubber stopper. The reaction may be initiated by addition of acell homogenate or, preferably a preparation of isolated mitochondria,via injection through the rubber stopper. In the assay described here,for example, a defect in complex IV activity may be correlated with anenzyme recognition site. This assay, or others based on similarprinciples, may permit correlation of mitochondrial respiratory activitywith mitochondria membrane permeability, which may be determinedaccording to other assays described herein.

[0078] The following examples are offered by way of illustration, andnot by way of limitation.

EXAMPLES Example 1

[0079]

[0080] STEP 1: Coupling Bromoacetic Acid on to Wang Resin

[0081] Polystyrene Wang resin (10.0 g, 1.25 mmol/g) was shaken at roomtemperature with bromoacetic acid (8.68 g, 62.5 mmol),diisopropylcarbodiimide (DIC) (9.79 ml, 62.5 mmol) and4-dimethylaminopyridine (DMAP) (100 mg) in DMF (60 ml) in apolypropylene bottle for 4 hours. The resin was collected via vacuumfiltration using a 50 ml polypropylene syringe fitted with apolyethylene frit, and washed with DMF (3×40 ml), methanol (3×40 ml),DMF (3×40 ml), methanol (3×40 ml), DCM (3×40 ml), methanol (3×40 ml),and air dried. The resulted bromoacetate resin 1 (12.0 g) was used inthe next step without further analysis.

[0082] STEP 2: Displacement Reaction with Amino Esters

[0083] a. Bromoacetate resin 1 (4.0 g) was shaken with glycine t-butylester HOAc salt (3.82 g, 20.0 mmol) and diisopropylethylamine (DIEA)(7.2 ml, 75 mmol) in DMSO (13 ml) in a 20 ml polypropylene syringefitted with a polyethylene frit at room temperature for 24 hours. Theresin was washed with DMF (3×20 ml), methanol (3×20 ml), DMF (3×20 ml),methanol (3×20 ml), DCM (3×20 ml), methanol (3×20 ml), and air dried.The resulted resin 2A was used in the next step without furtheranalysis.

[0084] b. Bromoacetate resin 1 (4.0 g) was treated with aspartic aciddi-t-butyl ester HCl salt in the same manner as described in 2a. Theresulted resin 2B was used in the next step without further analysis.

[0085] c. Bromoacetate resin 1 (4.0 g) was treated with glutaric aciddi-t-butyl ester HCl salt in the same manner as described in 2a. Theresulted resin 2C was used in the next step without further analysis.

[0086] STEP 3: Coupling Reaction with Nitrobenzoic Acids (A=Direct bond)

[0087] a. One-third of resin 2A was shaken with 2-nitrobenzoic acid(1.16 g, 6.9 mmol), DIEA (2.0 ml, 11.5 mmol), andbromo-(tris-pyrrolidino)phosphonium hexafluorophasphate(PyBrop) (3.26 g,7.0 mmol) in DMF (10 ml) at room temperature overnight. The resin waswashed with DMF (3×10 ml). The reaction was repeated to ensure completecoupling. The resin was washed with DMF (3×10 ml), methanol (3×10 ml),DMF (3×10 ml), methanol (3×10 ml), DCM (3×10 ml), mathanol (3×10 ml),and air dried. A small sample (˜50 mg) of the resulted resin 3AA wastreated with TFA/water (95/5, 1.0 ml) for 1 hour at room temperature.The solution was collected via filtration. The resin was washed withacetic acid (3×1 ml). The combined solution was lyophilized. The residuewas analyzed by 1H NMR and mass spectrometry. ¹H NMR (CD₃OD)_(—)8.25(m,1H), 7.82(m, 1H), 7.72(m, 1H), 7.54(m, 1H), 4.38(s, 2H), 4.07(s, 2H). MScalcd. for C₁₁H₁₀N₂O₇: 282.05, found: 281(M—H).

[0088] b. One-third of resin 2A was reacted with 3-nitrobenzoic acid inthe same manner as described in 3a to yield resin 3AB. ¹H NMR (CD₃OD)_(—)8.37(m, 1H), 8.32(m, 1H), 7.84(m, 1H), 7.74(m, 1H), 4.31(s, 2H),4.12(s, 2H). MS calcd. for C₁₁H₁₀N₂O₇: 282.05, found: 281(M—H).

[0089] c. One third of resin 2A was reacted with 4-nitrobenzoic acid inthe same manner as described in 3a to yield resin 3AC. ¹H NMR (CD₃OD)_(—)8.33(d, 2H), 7.67(d, 2H), 4.31(s, 2H), 4.09(s, 2H). MS calcd. forC₁₁H₁₀N₂O₇: 282.05, found: 281(M—H).

[0090] d. One-third of resin 2B was reacted with 2-nitrobenzoic acid inthe same manner as described in 3a to yield resin 3BA. MS calcd. forC₁₃H₁₂N₂O₉: 340.05, found: 339(M—H).

[0091] e. One-third of resin 2B was reacted with 3-nitrobenzoic acid inthe same manner as described in 3a to yield resin 3BB. MS calcd. forC₁₃H₁₂N₂O₉: 340.05, found: 339(M—H).

[0092] f. One-third of resin 2B was reacted with 4-nitrobenzoic acid inthe same manner as described in 3a to yield resin 3BC. MS calcd. forC₁₃H₁₂N₂O₉: 340.05, found: 339(M—H).

[0093] g. One-third for resin 2C was reacted with 2-nitrobenzoic acid inthe same manner as described in 3a to yield resin 3CA. MS calcd. forC₁₄H₁₄N₂O₉: 354.07, found: 353(M—H).

[0094] h. One-third of resin 2C was shaken with 3-nitrobenzoic acid inthe same manner as described in 3a to yield resin 3CB. MS calcd. forC₁₄H₁₄N₂O₉: 354.07, found: 353(M—H).

[0095] i. One-third of resin 2C was shaken with 4-nitrobenzoic acid inthe same manner as described in 3a to yield resin 3CC. MS calcd. forC₁₄H₁₄N₂O₉: 354.07, found: 353(M—H).

[0096] STEP 4. Reduction of Nitro Groups to Amines

[0097] a. Resin 3AA was shaken with tin dichloride dihydrate (2.0 M, 20ml) in DMF at room temperature overnight. The resin was washed with DMF(5×10 ml), methanol (3×10 ml), DMF (3×10 ml), methanol (3×10 ml), DCM(3×10 ml), methanol (3×10 ml), and air dried. A small sample (˜50 mg) ofthe resulting resin 4AA was treated with TFA/water (95/5, 1.0 ml) for 1hour at room temperature. The solution was collected via filtration. Theresin was washed with acetic acid (3×1 ml). The combined solution waslyophilized. The residue was analyzed by mass spectrometry. MS calcd.for C₁₁H₁₂N₂O₅: 252.07, found: 251(M—H).

[0098] b. Resin 3AB was treated in the same manner as described in 4a toyield 4AB. MS calcd. for C₁₁H₁₂N₂O₅: 252.07, found: 251(M—H).

[0099] c. Resin 3AC was treated in the same manner as described in 4a toyield 4AC. MS calcd. for C₁₁H₁₂N₂O₅: 252.07, found: 251(M—H).

[0100] d. Resin 3BA was treated in the same manner as described in 4a toyield 4BA. MS calcd. for C₁₂H₁₄N₂O₅: 310.08, found: 309(M—H).

[0101] e. Resin 3BB was treated in the same manner as described in 4a toyield 4BB. MS calcd. for C₁₂H₁₄N₂O₅: 310.08, found: 309(M—H).

[0102] f. Resin 3BC was treated in the same manner as described in 4a toyield 4BC. MS calcd. for C₁₂H₁₄N₂O₅: 310.08, found 309(M—H).

[0103] g. Resin 3CA was treated in the same manner as described in 4a toyield 4CA. MS calcd. for C₁₂H₁₄N₂O₅: 324.10, found 323(M—H).

[0104] h. Resin 3CB was treated in the same manner as described in 4a toyield 4CB. MS calcd. for C₁₂H₁₄N₂O₅: 324.10, found 323(M—H).

[0105] i. Resin 3CC was treated in the same manner as described in 4a toyield 4CC. MS calcd. for C₁₂H₁₄N₂O₅: 324.10, found 323(M—H).

[0106] STEP 5. Coupling of Carboxylic Acids on to Resin and TFA Cleavage

[0107] a. Resin 4AA was divided into 7 equal portions.

[0108] Portion A was shaken with acetic anhydride (A) (0.24 ml, 2.5mmol), DIEA (0.87 ml, 5.0 mmol) and DMAP (10 mg) in DMF (5.0 ml) at roomtemperature overnight. The resin was washed with DMF (3×5 ml), methanol(3×5 ml), DMF (3×5 ml), methanol (3×5 ml), DCM (3×5 ml), methanol (3×5ml), and air dried. The resulted resin 5AAA was treated with TFA/water(95/5, 3.0 ml) for 1 hour at room temperature. The solution wascollected via filtration. The resin was washed with acetic acid (3×5ml). The combined was lyophilized to give the desired product 6AAA. Itspurity and identity were assessed using HPLC-MS spectrometry.

[0109] Portion B was shaken with benzoic acid (B) (0.31 g, 2.5 mmol),DIC (0.47 ml, 3.0 mmol), DIEA (0.87 ml, 5.0 mmol) and DMAP (10 mg) inDMF (5.0 ml) at room temperature overnight. The resin was washed withDMF (3×5 ml), methanol (3×5 ml), DMF (3×5 ml), methanol (3×5 ml), DCM(3×5 ml), methanol (3×5 ml), and air dried. The resulting resin 5AAB wastreated with TFA/water (95/5, 3.0 ml) for 1 hour at room temperature.The solution was collected via filtration. The resin was washed withacetic acid (3×5 ml). The combined solution was lyophilized to give thedesired product 6AAB. Its purity and identity were assessed usingHPLC-MS spectrometry.

[0110] Portion C was treated with decanoic acid (C) in the same manneras described for Portion B.

[0111] Portion D was treated with glutaric anhydride (D) in the samemanner as described for Portion A.

[0112] Portion E was treated with heptanoyl chloride (E) in the samemanner as described for Portion A.

[0113] Portion F was treated with heptanoyl chloride (F) in the samemanner as described for Portion A.

[0114] Portion G was treated with heptanoyl chloride (G) in the samemanner as described for Portion A.

[0115] b. Resin 4AB, 4AC, 4BA, 4BB, 4BC, 4CA, 4CB, and 4CC were treatedusing the same procedure as described in Step 5a.

[0116] The compounds made according to the above procedures aresummarized in the following Table 1. In Table 1, it should be noted thatcompounds are identified with three-letter codes. Within thesethree-letter codes, the first letter codes for the first componentpiece, the second letter codes for the second component piece, and thethird letter codes for third component piece. Such first, second andthird component pieces are identified in the reaction scheme presentedabove in this example. TABLE 1 REPRESENTATIVE COMPOUNDS (R₁ OF STRUCTURE(I) = HYDROXY) MW First Second Third MW found Cpd. No. ComponentComponent Component Formula Calcd (M − H) 6AAA glycine t-butyl2-nitrobenzoic acetic C₁₃H₁₄N₂O₆ 294.3 293 ester (A) acid (A) anhydride(A) 6AAB glycine t-butyl 2-nitrobenzoic benzoic acid C₁₈H₁₆N₂O₆ 356.3355 ester (A) acid (A) (B) 6AAC glycine t-butyl 2-nitrobenzoic decanoicacid C₂₁H₃₀N₂O₆ 406.5 405 ester (A) acid (A) (C) 6AAD glycine t-butyl2-nitrobenzoic glutaric C₁₆H₁₈N₂O₈ 366.5 365 ester (A) acid (A)anhydride (D) 6AAE glycine t-butyl 2-nitrobenzoic heptanoyl C₁₈H₂₄N₂O₆364.4 363 ester (A) acid (A) chloride (E) 6AAF glycine t-butyl2-nitrobenzoic methyl C₂₂H₃₀N₂O₈ 450.5 449 ester (A) acid (A) sebacoylchloride (F) 6AAG glycine t-butyl 2-nitrobenzoic methyl C₂₀H₂₆N₂O₈ 422.4421 ester (A) acid (A) suberyl chloride (G) 6ABA glycine t-butyl3-nitrobenzoic acetic C₁₃H₁₄N₂O₆ 294.3 293 ester (A) acid (B) anhydride(A) 6ABB glycine t-butyl 3-nitrobenzoic benzoic acid C₁₈H₁₆N₂O₆ 356.3355 ester (A) acid (B) (B) 6ABC glycine t-butyl 3-nitrobenzoic decanoicacid C₂₁H₃₀N₂O₆ 406.5 405 ester (A) acid (B) (C) 6ABD glycine t-butyl3-nitrobenzoic glutaric C₁₆H₁₈N₂O₈ 366.3 365 ester (A) acid (B)anhydride (D) 6ABE glycine t-butyl 3-nitrobenzoic heptanoyl C₁₈H₂₄N₂O₆364.4 363 ester (A) acid (B) chloride (E) 6ABF glycine t-butyl3-nitrobenzoic methyl C₂₂H₃₀N₂O₈ 450.5 449 ester (A) acid (B) sebacoylchloride (F) 6ABG glycine t-butyl 3-nitrobenzoic methyl C₂₀H₂₆N₂O₈ 422.4421 ester (A) acid (B) suberyl chloride (G) 6ACA glycine t-butyl4-nitrobenzoic acetic C₁₃H₁₄N₂O₆ 294.3 293 ester (A) acid (C) anhydride(A) 6ACB glycine t-butyl 4-nitrobenzoic benzoic acid C₁₈H₁₆N₂O₆ 356.3355 ester (A) acid (C) (B) 6ACC glycine t-butyl 4-nitrobenzoic decanoicacid C₂₁H₃₀N₂O₆ 406.5 405 ester (A) acid (C) (C) 6ACD glycine t-butyl4-nitrobenzoic glutaric C₁₆H₁₈N₂O₈ 366.3 365 ester (A) acid (C)anhydride (D) 6ACE glycine t-butyl 4-nitrobenzoic heptanoyl C₁₈H₂₄N₂O₆364.4 363 ester (A) acid (C) chloride (E) 6ACF glycine t-butyl4-nitrobenzoic methyl C₂₂H₃₀N₂O₈ 450.5 449 ester (A) acid (C) sebacoylchloride (F) 6ACG glycine t-butyl 4-nitrobenzoic methyl C₂₀H₂₆N₂O₈ 422.4421 ester (A) acid (C) suberyl chloride (G) 6BAA aspartic acid2-nitrobenzoic acetic C₁₅H₁₆N₂O₈ 352.3 351 di-t-butyl ester acid (A)anhydride (A) (B) 6BAB aspartic acid 2-nitrobenzoic benzoic acidC₂₀H₁₈N₂O₈ 414.4 413 di-t-butyl ester acid (A) (B) (B) 6BAC asparticacid 2-nitrobenzoic decanoic acid C₂₃H₃₂N₂O₈ 464.5 464 di-t-butyl esteracid (A) (C) (B) 6BAD aspartic acid 2-nitrobenzoic glutaric C₁₈H₂₀N₂O₁₀424.4 423 di-t-butyl ester acid (A) anhydride (D) (B) 6BAE aspartic acid2-nitrobenzoic heptanoyl C₂₀H₂₅N₂O₈ 422.4 421 di-t-butyl ester acid (A)chloride (E) (B) 6BAF aspartic acid 2-nitrobenzoic methyl C₂₄H₃₂N₂O₁₀508.5 508 di-t-butyl ester acid (A) sebacoyl (B) chloride (F) 6BAGaspartic acid 2-nitrobenzoic methyl C₂₂H₂₈N₂O₁₀ 480.5 479 di-t-butylester acid (A) suberyl (B) chloride (G) 6BBA aspartic acid3-nitrobenzoic acetic C₁₅H₁₆N₂O₈ 352.3 351 di-t-butyl ester acid (B)anhydride (A) (B) 6BBB aspartic acid 3-nitrobenzoic benzoic acidC₂₀H₁₈N₂O₈ 414.4 413 di-t-butyl ester acid (B) (B) (B) 6BBC asparticacid 3-nitrobenzoic decanoic acid C₂₃H₃₂N₂O₈ 464.5 423 di-t-butyl esteracid (B) (C) (B) 6BBD aspartic acid 3-nitrobenzoic glutaric C₁₈H₂₀N₂O₁₀424.4 423 di-t-butyl ester acid (B) anhydride (D) (B) 6BBE aspartic acid3-nitrobenzoic heptanoyl C₂₀H₂₆N₂O₈ 422.4 421 di-t-butyl ester acid (B)chloride (E) (B) 6BBF aspartic acid 3-nitrobenzoic methyl C₂₄H₃₂N₂O₁₀508.5 508 di-t-butyl ester acid (B) sebacoyl (B) chloride (F) 6BBGaspartic acid 3-nitrobenzoic methyl C₂₂H₂₈N₂O₁₀ 480.5 479 di-t-butylester acid (B) suberyl (B) chloride (G) 6BCA aspartic acid4-nitrobenzoic acetic C₁₅H₁₆N₂O₈ 352.3 351 di-t-butyl ester acid (C)anhydride (A) (B) 6BCB aspartic acid 4-nitrobenzoic benzoic acidC₂₀H₁₈N₂O₈ 414.4 413 di-t-butyl ester acid (C) (B) (B) 6BCC asparticacid 4-nitrobenzoic decanoic acid C₂₃H₃₂N₂O₈ 464.5 464 di-t-butyl esteracid (C) (C) (B) 6BCD aspartic acid 4-nitrobenzoic glutaric C₁₈H₂₀N₂O₁₀424.4 423 di-t-butyl ester acid (C) anhydride (D) (B) 6BCE aspartic acid4-nitrobenzoic heptanoyl C₂₀H₂₆N₂O₈ 422.4 421 di-t-butyl ester acid (C)chloride (E) (B) 6BCF aspartic acid 4-nitrobenzoic methyl C₂₄H₃₂N₂O₁₀508.5 508 di-t-butyl ester acid (C) sebacoyl (B) chloride (F) 6BCGaspartic acid 4-nitrobenzoic methyl C₂₂H₂₈N₂O₁₀ 480.5 479 di-t-butylester acid (C) suberyl (B) chloride (G) 6CAA glutaric acid2-nitrobenzoic acetic C₁₆H₁₈N₂O₈ 366.3 365 di-t-butyl ester acid (A)anhydride (A) (C) 6CAB glutaric acid 2-nitrobenzoic benzoic acidC₂₁H₂₀N₂O₈ 428.4 427 di-t-butyl ester acid (A) (B) (C) 6CAC glutaricacid 2-nitrobenzoic decanoic acid C₂₄H₃₄N₂O₈ 478.5 478 di-t-butyl esteracid (A) (C) (C) 6CAD glutaric acid 2-nitrobenzoic glutaric C₁₉H₂₂N₂O₁₀438.4 437 di-t-butyl ester acid (A) anhydride (D) (C) 6CAE glutaric acid2-nitrobenzoic heptanoyl C₂₁H₂₈N₂O₈ 436.6 435 di-t-butyl ester acid (A)chloride (E) (C) 6CAF glutaric acid 2-nitrobenzoic methyl C₂₅H₃₄N₂O₁₀522.6 522 di-t-butyl ester acid (A) sebacoyl (C) chloride (F) 6CAGglutaric acid 2-nitrobenzoic methyl C₂₃H₃₀N₂O₁₀ 494.5 493 di-t-butylester acid (A) suberyl (C) chloride (G) 6CBA glutaric acid3-nitrobenzoic acetic C₁₆H₁₈N₂O₈ 366.3 365 di-t-butyl ester acid (B)anhydride (A) (C) 6CBB glutaric acid 3-nitrobenzoic benzoic acidC₂₁H₂₀N₂O₈ 428.4 427 di-t-butyl ester acid (B) (B) (C) 6CBC glutaricacid 3-nitrobenzoic decanoic acid C₂₄H₃₄N₂O₈ 478.5 478 di-t-butyl esteracid (B) (C) (C) 6CBD glutaric acid 3-nitrobenzoic glutaric C₁₉H₂₂N₂O₁₀438.4 437 di-t-butyl ester acid (B) anhydride (D) (C) 6CBE glutaric acid3-nitrobenzoic heptanoyl C₂₁H₂₈N₂O₈ 436.5 435 di-t-butyl ester acid (B)chloride (E) (C) 6CBF glutaric acid 3-nitrobenzoic methyl C₂₅H₃₄N₂O₁₀522.6 522 di-t-butyl ester acid (B) sebacoyl (C) chloride (F) 6CBGglutaric acid 3-nitrobenzoic methyl C₂₃H₃₀N₂O₁₀ 494.5 493 di-t-butylester acid (B) suberyl (C) chloride (G) 6CCA glutaric acid4-nitrobenzoic acetic C₁₆H₁₈N₂O₈ 366.3 365 di-t-butyl ester acid (C)anhydride (A) (C) 6CCB glutaric acid 4-nitrobenzoic benzoic acidC₂₁H₂₀N₂O₈ 428.4 427 di-t-butyl ester acid (C) (B) (C) 6CCC glutaricacid 4-nitrobenzoic decanoic acid C₂₄H₃₄N₂O₈ 478.5 478 di-t-butyl esteracid (C) (C) (C) 6CCD glutaric acid 4-nitrobenzoic glutaric C₁₉H₂₂N₂O₁₀438.4 437 di-t-butyl ester acid (C) anhydride (D) (C) 6CCE glutaric acid4-nitrobenzoic heptanoyl C₂₁H₂₈N₂O₈ 436.5 435 di-t-butyl ester acid (C)chloride (E) (C) 6CCF glutaric acid 4-nitrobenzoic methyl C₂₅H₃₄N₂O₁₀522.6 522 di-t-butyl ester acid (C) sebacoyl (C) chloride (F) 6CCGglutaric acid 4-nitrobenzoic methyl C₂₃H₃₀N₂O₁₀ 494.5 493 di-t-butylester acid (C) suberyl (C) chloride (G)

Example 2 Synthesis of Representative Compounds of Structure (I)

[0117] Using the same procedures as illustrated in Example 1, additionalrepresentative compounds were prepared using glycine methyl ester (A) orglycinamide (B) as the first component. The corresponding compounds arelisted below in Table 2. TABLE 2 REPRESENTATIVE COMPOUNDS (R₁ OFSTRUCTURE (I) = METHOXY OR AMINO) MW First Second Third MW found Cpd.No. Component Component Component Formula Calcd (M − H) 7AAA glycinemethyl 3-nitrobenzoic glutaric C₁₇H₂₀N₂O₈ 380.4 381 ester (A) acid (A)anhydride (A) 7AAB glycine methyl 3-nitrobenzoic heptanoyl C₁₉H₂₆N₂O₆378.4 379 ester (A) acid (A) chloride (B) 7AAC glycine methyl3-nitrobenzoic decanoic acid C₂₂H₃₂N₂O₆ 420.5 421 ester (A) acid (A) (C)7AAD glycine methyl 3-nitrobenzoic methyl C₂₁H₂₈N₂O₈ 436.5 437 ester (A)acid (A) suberyl chloride (D) 7AAE glycine methyl 3-nitrobenzoic methylC₂₃H₃₂N₂O₈ 464.5 465 ester (A) acid (A) sebacoyl chloride (E) 7ABAglycine methyl 4-nitrobenzoic glutaric C₁₇H₂₀N₂O₈ 380.4 381 ester (A)acid (B) anhydride (A) 7ABB glycine methyl 4-nitrobenzoic heptanoylC₁₉H₂₆N₂O₆ 378.4 379 ester (A) acid (B) chloride (B) 7ABC glycine methyl4-nitrobenzoic decanoic acid C₂₂H₃₂N₂O₆ 420.5 421 ester (A) acid (B) (C)7ABD glycine methyl 4-nitrobenzoic methyl C₂₁H₂₈N₂O₈ 436.5 437 ester (A)acid (B) suberyl chloride (D) 7ABE glycine methyl 4-nitrobenzoic methylC₂₃H₃₂N₂O₈ 464.5 465 ester (A) acid (B) sebacoyl chloride (E) 7BAAglycinamide 3-nitrobenzoic glutaric C₁₆H₁₉N₃O₇ 365.3 366 (B) acid (A)anhydride (A) 7BAB glycinamide 3-nitrobenzoic heptanoyl C₁₈H₂₅N₃O₅ 363.4364 (B) acid (A) chloride (B) 7BAC glycinamide 3-nitrobenzoic decanoicacid C₂₁H₃₁N₃O₅ 405.5 406 (B) acid (A) (C) 7BAD glycinamide3-nitrobenzoic methyl C₂₀H₂₇N₃O₇ 421.4 422 (B) acid (A) suberyl chloride(D) 7BAE glycinamide 3-nitrobenzoic methyl C₂₂H₃₁N₃O₇ 449.5 450 (B) acid(A) sebacoyl chloride (E) 7BBA glycinamide 4-nitrobenzoic glutaricC₁₆H₁₉N₃O₇ 365.3 366 (B) acid (B) anhydride (A) 7BBB glycinamide4-nitrobenzoic heptanoyl C₁₈H₂₅N₃O₅ 363.4 364 (B) acid (B) chloride (B)7BBC glycinamide 4-nitrobenzoic decanoic acid C₂₁H₃₁N₃O₅ 405.5 406 (B)acid (B) (C) 7BBD glycinamide 4-nitrobenzoic methyl C₂₀H₂₇N₃O₇ 421.4 422(B) acid (B) suberyl chloride (D) 7BBE glycinamide 4-nitrobenzoic methylC₂₂H₃₁N₃O₇ 449.5 450 (B) acid (B) sebacoyl chloride (E)

Example 3 Synthesis of Representative Compounds of Structure (I)

[0118] Using the same procedures as illustrated in Example 1, additionalrepresentative compounds were prepared using alanine benzyl ester (A),valine benzyl ester (B), leucine benzyl ester (C) or phenylalaninebenzyl ester (D) as the first component. The corresponding products arelisted below in Table 3. TABLE 3 REPRESENTATIVE COMPOUNDS (R₁ OFSTRUCTURE (I) = BNO) MW First Second Third MW found Cpd. No. ComponentComponent Component Formula Calcd (M − H) 8AAA alanine benzyl3-nitrobenzoic glutaric C₂₆H₃₀N₂O₈ 498.5 499 ester (A) acid (A)anhydride (A) 8AAB alanine benzyl 3-nitrobenzoic heptanoyl C₂₈H₃₆N₂O₆496.6 497 ester (A) acid (A) chloride (B) 8AAC alanine benzyl3-nitrobenzoic decanoic acid C₃₁H₄₂N₂O₆ 538.7 539 ester (A) acid (A) (C)8AAD alanine benzyl 3-nitrobenzoic methyl C₃₀H₃₈N₂O₈ 554.6 555 ester (A)acid (A) suberyl chloride (D) 8AAE alanine benzyl 3-nitrobenzoic methylC₃₂H₄₂N₂O₈ 582.7 583 ester (A) acid (A) sebacoyl chloride (E) 8ABAalanine benzyl 4-nitrobenzoic glutaric C₂₀H₂₆N₂O₈ 422.4 423 ester (A)acid (B) anhydride (A) 8ABB alanine benzyl 4-nitrobenzoic heptanoylC₂₂H₃₂N₂O₆ 420.5 421 ester (A) acid (B) chloride (B) 8ABC alanine benzyl4-nitrobenzoic decanoic acid C₂₅H₃₈N₂O₆ 462.6 463 ester (A) acid (B) (C)8ABD alanine benzyl 4-nitrobenzoic methyl C₂₄H₃₄N₂O₈ 478.5 479 ester (A)acid (B) suberyl chloride (D) 8ABE alanine benzyl 4-nitrobenzoic methylC₂₆H₃₈N₂O₈ 506.6 507 ester (A) acid (B) sebacoyl chloride (E) 8BAAvaline benzyl 3-nitrobenzoic glutaric C₂₇H₃₂N₂O₈ 512.6 513 ester (B)acid (A) anhydride (A) 8BAB valine benzyl 3-nitrobenzoic heptanoylC₂₉H₃₈N₂O₆ 510.6 511 ester (B) acid (A) chloride (B) 8BAC valine benzyl3-nitrobenzoic decanoic acid C₃₂H₄₄N₂O₆ 552.7 553 ester (B) acid (A) (C)8BAD valine benzyl 3-nitrobenzoic methyl C₃₁H₄₀N₂O₈ 568.7 569 ester (B)acid (A) suberyl chloride (D) 8BAE valine benzyl 4-nitrobenzoic methylC₃₃H₄₄N₂O₈ 596.7 597 ester (B) acid (B) sebacoyl chloride (E) 8BBAvaline benzyl 4-nitrobenzoic glutaric C₂₀H₂₆N₂O₈ 422.4 423 ester (B)acid (B) anhydride (A) 8BBB valine benzyl 4-nitrobenzoic heptanoylC₂₂H₃₂N₂O₆ 420.5 421 ester (B) acid (B) chloride (B) 8BBC valine benzyl4-nitrobenzoic decanoic acid C₂₅H₃₈N₂O₆ 462.6 463 ester (B) acid (B) (C)8BBD valine benzyl 4-nitrobenzoic methyl C₂₄H₃₄N₂O₈ 478.5 479 ester (B)acid (B) suberyl chloride (D) 8BBE valine benzyl 4-nitrobenzoic methylC₂₆H₃₈N₂O₈ 506.6 507 ester (B) acid (B) sebacoyl chloride (E) 8CAAleucine benzyl 3-nitrobenzoic glutaric C₂₇H₃₂N₂O₈ 512.6 513 ester (C)acid (A) anhydride (A) 8CAB leucine benzyl 3-nitrobenzoic heptanoylC₂₉H₃₈N₂O₆ 510.6 511 ester (C) acid (A) chloride (B) 8CAC leucine benzyl3-nitrobenzoic decanoic acid C₃₂H₄₄N₂O₆ 552.7 553 ester (C) acid (A) (C)8CAD leucine benzyl 3-nitrobenzoic methyl C₃₁H₄₀N₂O₈ 568.7 569 ester (C)acid (A) suberyl chloride (D) 8CAE leucine benzyl 3-nitrobenzoic methylC₃₃H₄₄N₂O₈ 596.7 597 ester (C) acid (A) sebacoyl chloride (E) 8CBAleucine benzyl 4-nitrobenzoic glutaric C₂₃H₂₄N₂O₈ 456.6 457 ester (C)acid (B) anhydride (A) 8CBB leucine benzyl 4-nitrobenzoic heptanoylC₂₅H₃₀N₂O₆ 454.5 455 ester (C) acid (B) chloride (B) 8CBC leucine benzyl4-nitrobenzoic decanoic acid C₂₈H₃₆N₂O₆ 496.6 497 ester (C) acid (B) (C)8CBD leucine benzyl 4-nitrobenzoic methyl C₂₇H₃₂N₂O₈ 512.6 513 ester (C)acid (B) suberyl chloride (D) 8CBE leucine benzyl 4-nitrobenzoic methylC₂₉H₃₆N₂O₈ 540.6 541 ester (C) acid (B) sebacoyl chloride (E) 8DBAphenylalanine 4-nitrobenzoic glutaric C₃₀H₃₀N₂O₈ 546.6 547 benzyl esteracid (B) anhydride (A) (D) 8DBB phenylalanine 4-nitrobenzoic heptanoylC₃₂H₃₆N₂O₆ 544.6 545 benzyl ester acid (B) chloride (B) (D) 8DBCphenylalanine 4-nitrobenzoic decanoic acid C₃₅H₄₂N₂O₆ 586.7 587 benzylester acid (B) (C) (D) 8DBD phenylalanine 4-nitrobenzoic methylC₃₄H₃₈N₂O₈ 602.7 603 benzyl ester acid (B) suberyl (D) chloride (D) 8DBEphenylalanine 4-nitrobenzoic methyl C₃₆H₄₂N₂O₈ 630.7 631 benzyl esteracid (B) sebacoyl (D) chloride (E)

Example 4 Synthesis of Representative Compounds of Structure (I)

[0119] Each of the compounds of Example 3 was divided into two portions.One portion was treated with hydrogen (10 psi) in the presence of 10%Palladium on activated carbon (15 mg) in acetic acid/methanol (1/4, 5ml) at room temperature overnight. The Pd/C was removed by filtrationand washed with acetic acid (3×5 ml). This resulted in the conversion ofthe benzyl ester (R₁=BnO) to the corresponding acid (R₁=OH). Theresulting compounds are summarized in Table 4. TABLE 4 REPRESENTATIVECOMPOUNDS (R₁ OF STRUCTURE (I) = OH) MW First Second Third MW found Cpd.No. Component Component Component Formula Calcd (M − H) 9AAA alaninebenzyl 3-nitrobenzoic glutaric C₁₇H₂₀N₂O₈ 380.4 381 ester (A) acid (A)anhydride (A) 9AAB alanine benzyl 3-nitrobenzoic heptanoyl C₁₉H₂₆N₂O₆378.4 379 ester (A) acid (A) chloride (B) 9AAC alanine benzyl3-nitrobenzoic decanoic acid C₂₂H₃₂N₂O₆ 420.5 421 ester (A) acid (A) (C)9AAD alanine benzyl 3-nitrobenzoic methyl C₂₁H₂₈N₂O₈ 436.5 437 ester (A)acid (A) suberyl chloride (D) 9AAE alanine benzyl 3-nitrobenzoic methylC₂₃H₃₂N₂O₈ 464.5 465 ester (A) acid (A) sebacoyl chloride (E) 9ABAalanine benzyl 4-nitrobenzoic glutaric C₂₄H₂₆N₂O₈ 470.5 471 ester (A)acid (B anhydride (A) 9ABB alanine benzyl 4-nitrobenzoic heptanoylC₂₆H₃₂N₂O₆ 468.5 469 ester (A) acid (B chloride (B) 9ABC alanine benzyl4-nitrobenzoic decanoic acid C₂₉H₃₈N₂O₆ 510.6 511 ester (A) acid (B (C)9ABD alanine benzyl 4-nitrobenzoic methyl C₂₈H₃₄N₂O₈ 526.6 527 ester (A)acid (B suberyl chloride (D) 9ABE alanine benzyl 4-nitrobenzoic methylC₃₀H₃₈N₂O₈ 554.6 555 ester (A) acid (B sebacoyl chloride (E) 9BAA valinebenzyl 3-nitrobenzoic glutaric C₁₇H₂₀N₂O₈ 380.4 381 ester (B) acid (A)anhydride (A) 9BAB valine benzyl 3-nitrobenzoic heptanoyl C₁₉H₂₆N₂O₆378.4 379 ester (B) acid (A) chloride (B) 9BAC valine benzyl3-nitrobenzoic decanoic acid C₂₂H₃₂N₂O₆ 420.5 421 ester (B) acid (A) (C)9BAD valine benzyl 3-nitrobenzoic methyl C₂₁H₂₈N₂O₈ 436.5 437 ester (B)acid (A) suberyl chloride (D) 9BAE valine benzyl 3-nitrobenzoic methylC₂₃H₃₂N₂O₈ 464.5 465 ester (B) acid (A) sebacoyl chloride (E) 9BBAvaline benzyl 4-nitrobenzoic glutaric C₂₄H₂₆N₂O₈ 470.5 471 ester (B)acid (B) anhydride (A) 9BBB valine benzyl 4-nitrobenzoic heptanoylC₂₆H₃₂N₂O₆ 468.5 469 ester (B) acid (B) chloride (B) 9BBC valine benzyl4-nitrobenzoic decanoic acid C₂₉H₃₈N₂O₆ 510.6 511 ester (B) acid (B) (C)9BBD valine benzyl 4-nitrobenzoic methyl C₂₈H₃₄N₂O₈ 526.6 527 ester (B)acid (B) suberyl chloride (D) 9BBE valine benzyl 4-nitrobenzoic methylC₃₀H₃₈N₂O₈ 554.6 555 ester (B) acid (B) sebacoyl chloride (E) 9CAAleucine benzyl 3-nitrobenzoic glutaric C₁₉H₂₄N₂O₈ 408.4 409 ester (C)acid (A) anhydride (A) 9CAB leucine benzyl 3-nitrobenzoic heptanoylC₂₁H₃₀N₂O₆ 406.5 407 ester (C) acid (A) chloride (B) 9CAC leucine benzyl3-nitrobenzoic decanoic acid C₂₄H₃₆N₂O₆ 448.6 449 ester (C) acid (A) (C)9CAD leucine benzyl 3-nitrobenzoic methyl C₂₃H₃₂N₂O₈ 464.5 465 ester (C)acid (A) suberyl chloride (D) 9CAE leucine benzyl 3-nitrobenzoic methylC₂₅H₃₆N₂O₈ 492.6 493 ester (C) acid (A) sebacoyl chloride (E) 9CBAleucine benzyl 4-nitrobenzoic glutaric C₂₆H₃₀N₂O₈ 498.5 499 ester (C)acid (B) anhydride (A) 9CBB leucine benzyl 4-nitrobenzoic heptanoylC₂₈H₃₆N₂O₆ 496.6 497 ester (C) acid (B) chloride (B) 9CBC leucine benzyl4-nitrobenzoic decanoic acid C₃₁H₄₂N₂O₆ 538.7 539 ester (C) acid (B) (C)9CBD leucine benzyl 4-nitrobenzoic methyl C₃₀H₃₈N₂O₈ 554.6 555 ester (C)acid (B) suberyl chloride (D) 9CBE leucine benzyl 4-nitrobenzoic methylC₃₂H₄₂N₂O₈ 582.7 583 ester (C) acid (B) sebacoyl chloride (E) 9DBAphenylalanine 4-nitrobenzoic glutaric C₁₉H₂₄N₂O₈ 408.4 409 benzyl esteracid (B) anhydride (A) (D) 9DBB phenylalanine 4-nitrobenzoic heptanoylC₂₁H₃₀N₂O₆ 406.5 407 benzyl ester acid (B) chloride (B) (D) 9DBCphenylalanine 4-nitrobenzoic decanoic acid C₂₄H₃₆N₂O₆ 448.6 449 benzylester acid (B) (C) (D) 9DBD phenylalanine 4-nitrobenzoic methylC₂₃H₃₂N₂O₈ 464.5 465 benzyl ester acid (B) suberyl (D) chloride (D) 9DBEphenylalanine 4-nitrobenzoic methyl C₂₅H₃₆N₂O₈ 492.6 493 benzyl esteracid (B) sebacoyl (D) chloride (E)

Example 5 Synthesis of Representative Compounds of Structure (I)

[0120] Using the same procedures as illustrated in Example 1, additionalrepresentative compounds were prepared using leucine benzyl ester (A) orphenylalanine benzyl ester (B) as the first component, and using varioussecond components to illustrate embodiments wherein the “A” moiety ofstructure (I) is other than a direct bond. The corresponding compoundsare listed below in Table 5. TABLE 5 REPRESENTATIVE COMPOUNDS (R₁ OFSTRUCTURE (I) = BNO) MW First Second Third MW found Cpd. No. ComponentComponent Component Formula Calcd (M − H) 10AAA Leucine 2-nitrophenyl-glutaric C₂₈H₃₄N₂O₈ 526.6 527 benzyl ester acetic acid (A) anhydride (A)(A) 10AAB Leucine 2-nitrophenyl- heptanoyl C₃₀H₄₀N₂O₆ 524.7 525 benzylester acetic acid (A) chloride (B) (A) 10AAC Leucine 2-nitrophenyl-decanoic acid C₃₃H₄₆N₂O₆ 566.7 567 benzyl ester acetic acid (A) (C) (A)10AAD Leucine 2-nitrophenyl- methyl C₃₂H₄₂N₂O₈ 582.7 583 benzyl esteracetic acid (A) suberyl (A) chloride (D) 10AAE Leucine 2-nitrophenyl-methyl C₃₄H₄₆N₂O₈ 610.7 611 benzyl ester acetic acid (A) sebacoyl (A)chloride (E) 10BAA Phenylalanine 2-nitrophenyl- glutaric C₃₁H₃₂N₂O₈560.6 561 benzyl ester acetic acid (A) anhydride (B) (A) 10BABPhenylalanine 2-nitrophenyl- heptanoyl C₃₃H₃₈N₂O₆ 558.7 559 benzyl esteracetic acid (A) chloride (B) (B) 10BAC Phenylalanine 2-nitrophenyl-decanoic acid C₃₆H₄₄N₂O₆ 600.8 601 benzyl ester acetic acid (A) (C) (B)10BAD Phenylalanine 2-nitrophenyl- methyl C₃₅H₄₀N₂O₈ 616.7 617 benzylester acetic acid (A) suberyl (B) chloride (D) 10BAE Phenylalanine2-nitrophenyl- methyl C₃₇H₄₄N₂O₈ 644.8 645 benzyl ester acetic acid (A)sebacoyl (B) chloride (E) 10ABA Leucine 3-nitrophenyl- glutaricC₂₈H₃₄N₂O₈ 526.6 527 benzyl ester acetic acid (B) anhydride (A) (A)10ABB Leucine 3-nitrophenyl- heptanoyl C₃₀H₄₀N₂O₆ 524.7 525 benzyl esteracetic acid (B) chloride (B) (A) 10ABC Leucine 3-nitrophenyl- decanoicacid C₃₃H₄₆N₂O₆ 566.7 567 benzyl ester acetic acid (B) (C) (A) 10ABDLeucine 3-nitrophenyl- methyl C₃₂H₄₂N₂O₈ 582.7 583 benzyl ester aceticacid (B) suberyl (A) chloride (D) 10ABE Leucine 3-nitrophenyl- methylC₃₄H₄₆N₂O₈ 610.7 611 benzyl ester acetic acid (B) sebacoyl (A) chloride(E) 10BBA Phenylalanine 3-nitrophenyl- glutaric C₃₁H₃₂N₂O₈ 560.6 561benzyl ester acetic acid (B) anhydride (B) (A) 10BBB Phenylalanine3-nitrophenyl- heptanoyl C₃₃H₃₈N₂O₆ 558.7 559 benzyl ester acetic acid(B) chloride (B) (B) 10BBC Phenylalanine 3-nitrophenyl- decanoic acidC₃₆H₄₄N₂O₆ 600.8 601 benzyl ester acetic acid (B) (C) (B) 10BBDPhenylalanine 3-nitrophenyl- methyl C₃₅H₄₀N₂O₈ 616.7 617 benzyl esteracetic acid (B) suberyl (B) chloride (D) 10BBE Phenylalanine3-nitrophenyl- methyl C₃₇H₄₄N₂O₈ 644.8 645 benzyl ester acetic acid (B)sebacoyl (B) chloride (E) 10ACA Leucine 4-nitrophenyl- glutaricC₂₈H₃₄N₂O₈ 526.6 527 benzyl ester acetic acid (C) anhydride (A) (A)10ACB Leucine 4-nitrophenyl- heptanoyl C₃₀H₄₀N₂O₆ 524.7 525 benzyl esteracetic acid (C) chloride (B) (A) 10ACC Leucine 4-nitrophenyl- decanoicacid C₃₃H₄₆N₂O₆ 566.7 567 benzyl ester acetic acid (C) (C) (A) 10ACDLeucine 4-nitrophenyl- methyl C₃₂H₄₂N₂O₈ 582.7 583 benzyl ester aceticacid (C) suberyl (A) chloride (D) 10ACE Leucine 4-nitrophenyl- methylC₃₄H₄₆N₂O₈ 610.7 611 benzyl ester acetic acid (C) sebacoyl (A) chloride(E) 10BCA Phenylalanine 4-nitrophenyl- glutaric C₃₁H₃₂N₂O₈ 560.6 561benzyl ester acetic acid (C) anhydride (B) (A) 10BCB Phenylalanine4-nitrophenyl- heptanoyl C₃₃H₃₈N₂O₆ 558.7 559 benzyl ester acetic acid(C) chloride (B) (B) 10BCC Phenylalanine 4-nitrophenyl- decanoic acidC₃₆H₄₄N₂O₆ 600.8 601 benzyl ester acetic acid (C) (C) (B) 10BCDPhenylalanine 4-nitrophenyl- methyl C₃₅H₄₀N₂O₈ 616.7 617 benzyl esteracetic acid (C) suberyl (B) chloride (D) 10BCE Phenylalanine4-nitrophenyl- methyl C₃₇H₄₄N₂O₈ 644.8 645 benzyl ester acetic acid (C)sebacoyl (B) chloride (E) 10ADA Leucine 2- glutaric C₂₈H₃₄N₂O₉ 542.6 543benzyl ester nitrophenoxy- anhydride (A) acetic acid (D) (A) 10ADDLeucine 2- methyl C₃₂H₄₂N₂O₉ 598.7 599 benzyl ester nitrophenoxy-suberyl (A) acetic acid (D) chloride (D) 10ADE Leucine 2- methylC₃₄H₄₆N₂O₉ 626.7 627 benzyl ester nitrophenoxy- sebacoyl (A) acetic acid(D) chloride (E) 10BDA Phenylalanine 2- glutaric C₃₁H₃₂N₂O₉ 576.6 577benzyl ester nitrophenoxy- anhydride (B) acetic acid (D) (A) 10BDBPhenylalanine 2- heptanoyl C₃₃H₃₈N₂O₇ 574.7 575 benzyl esternitrophenoxy- chloride (B) (B) acetic acid (D) 10BDC Phenylalanine 2-decanoic acid C₃₆H₄₄N₂O₇ 616.8 617 benzyl ester nitrophenoxy- (C) (B)acetic acid (D) 10AEA Leucine 3- glutaric C₂₈H₃₄N₂O₉ 542.6 543 benzylester nitrophenoxy- anhydride (A) acetic acid (E) (A) 10AEB Leucine 3-heptanoyl C₃₀H₄₀N₂O₇ 540.7 541 benzyl ester nitrophenoxy- chloride (B)(A) acetic acid (E) 10AEC Leucine 3- decanoic acid C₃₃H₄₆N₂O₇ 582.7 583benzyl ester nitrophenoxy- (C) (A) acetic acid (E) 10AED Leucine 3-methyl C₃₂H₄₂N₂O₉ 598.7 599 benzyl ester nitrophenoxy- suberyl (A)acetic acid (E) chloride (D) 10AEE Leucine 3- methyl C₃₄H₄₆N₂O₉ 626.7627 benzyl ester nitrophenoxy- sebacoyl (A) acetic acid (E) chloride (E)10BEA Phenylalanine 3- glutaric C₃₁H₃₂N₂O₉ 576.6 577 benzyl esternitrophenoxy- anhydride (B) acetic acid (E) (A) 10BEB Phenylalanine 3-heptanoyl C₃₃H₃₈N₂O₇ 574.7 575 benzyl ester nitrophenoxy- chloride (B)(B) acetic acid (E) 10BEC Phenylalanine 3- decanoic acid C₃₆H₄₄N₂O₇616.8 617 benzyl ester nitrophenoxy- (C) (B) acetic acid (E) 10BEDPhenylalanine 3- methyl C₃₅H₄₀N₂O₉ 632.7 633 benzyl ester nitrophenoxy-suberyl (B) acetic acid (E) chloride (D) 10BEE Phenylalanine 3- methylC₃₇H₄₄N₂O₉ 660.8 661 benzyl ester nitrophenoxy- sebacoyl (B) acetic acid(E) chloride (E) 10AFA Leucine 4- glutaric C₂₈H₃₄N₂O₉ 542.6 543 benzylester nitrophenoxy- anhydride (A) acetic acid (F) (A) 10AFB Leucine 4-heptanoyl C₃₀H₄₀N₂O₇ 540.7 541 benzyl ester nitrophenoxy- chloride (B)(A) acetic acid (F) 10AFC Leucine 4- decanoic acid C₃₃H₄₆N₂O₇ 582.7 583benzyl ester nitrophenoxy- (C) (A) acetic acid (F) 10AFD Leucine 4-methyl C₃₂H₄₂N₂O₉ 598.7 599 benzyl ester nitrophenoxy- suberyl (A)acetic acid (F) chloride (D) 10AFE Leucine 4- methyl C₃₄H₄₆N₂O₉ 626.7627 benzyl ester nitrophenoxy- sebacoyl (A) acetic acid (F) chloride (E)10BFA Phenylalanine 4- glutaric C₃₁H₃₂N₂O₉ 576.6 577 benzyl esternitrophenoxy- anhydride (B) acetic acid (F) (A) 10BFB Phenylalanine 4-heptanoyl C₃₃H₃₈N₂O₇ 574.7 575 benzyl ester nitrophenoxy- chloride (B)(B) acetic acid (F) 10BFC Phenylalanine 4- decanoic acid C₃₆H₄₄N₂O₇616.8 617 benzyl ester nitrophenoxy- (C) (B) acetic acid (F) 10BFDPhenylalanine 4- methyl C₃₅H₄₀N₂O₉ 632.7 633 benzyl ester nitrophenoxy-suberyl (B) acetic acid (F) chloride (D) 10BFE Phenylalanine 4- methylC₃₇H₄₄N₂O₉ 660.8 661 benzyl ester nitrophenoxy- sebacoyl (B) acetic acid(F) chloride (E) 10AGA Leucine 2- glutaric C₂₉H₃₄N₂O₈ 538.6 539 benzylester nitrocinnamic anhydride (A) acid (G) (A) 10AGB Leucine 2-heptanoyl C₃₁H₄₀N₂O₆ 536.7 537 benzyl ester nitrocinnamic chloride (B)(A) acid (G) 10AGC Leucine 2- decanoic acid C₃₄H₄₆N₂O₆ 578.7 579 benzylester nitrocinnamic (C) (A) acid (G) 10AGD Leucine 2- methyl C₃₃H₄₂N₂O₈594.7 595 benzyl ester nitrocinnamic suberyl (A) acid (G) chloride (D)10AGE Leucine 2- methyl C₃₅H₄₆N₂O₈ 622.8 623 benzyl ester nitrocinnamicsebacoyl (A) acid (G) chloride (E) 10BGA Phenylalanine 2- glutaricC₂₅H₂₆N₂O₈ 572.6 573 benzyl ester nitrocinnamic anhydride (B) acid (G)(A) 10BGB Phenylalanine 2- heptanoyl C₂₇H₃₂N₂O₆ 570.3 571 benzyl esternitrocinnamic chloride (B) (B) acid (G) 10BGC Phenylalanine 2- decanoicacid C₃₀H₃₈N₂O₆ 612.8 613 benzyl ester nitrocinnamic (C) (B) acid (G)10BGD Phenylalanine 2- methyl C₂₉H₃₄N₂O₈ 628.7 629 benzyl esternitrocinnamic suberyl (B) acid (G) chloride (D) 10BGE Phenylalanine 2-methyl C₃₁H₃₈N₂O₈ 656.8 657 benzyl ester nitrocinnamic sebacoyl (B) acid(G) chloride (E) 10AHA Leucine 3- glutaric C₂₉H₃₄N₂O₈ 538.6 539 benzylester nitrocinnamic anhydride (A) acid (H) (A) 10AHB Leucine 3-heptanoyl C₃₁H₄₀N₂O₆ 536.7 537 benzyl ester nitrocinnamic chloride (B)(A) acid (H) 10AHC Leucine 3- decanoic acid C₃₄H₄₆N₂O₆ 578.7 579 benzylester nitrocinnamic (C) (A) acid (H) 10AHD Leucine 3- methyl C₃₃H₄₂N₂O₈594.7 595 benzyl ester nitrocinnamic suberyl (A) acid (H) chloride (D)10AHE Leucine 3- methyl C₃₅H₄₆N₂O₈ 622.8 623 benzyl ester nitrocinnamicsebacoyl (A) acid (H) chloride (E) 10BHA Phenylalanine 3- glutaricC₃₂H₃₂N₂O₈ 572.6 573 benzyl ester nitrocinnamic anhydride (B) acid (H)(A) 10BHB Phenylalanine 3- heptanoyl C₃₄H₃₈N₂O₆ 570.7 571 benzyl esternitrocinnamic chloride (B) (B) acid (H) 10BHC Phenylalanine 3- decanoicacid C₃₇H₄₄N₂O₆ 612.8 613 benzyl ester nitrocinnamic (C) (B) acid (H)10BHD Phenylalanine 3- methyl C₃₆H₄₀N₂O₈ 628.7 629 benzyl esternitrocinnamic suberyl (B) acid (H) chloride (D) 10BHE Phenylalanine 3-methyl C₃₈H₄₄N₂O₈ 656.8 657 benzyl ester nitrocinnamic sebacoyl (B) acid(H) chloride (E) 10AIA Leucine 4- glutaric C₂₉H₃₄N₂O₈ 538.6 539 benzylester nitrocinnamic anhydride (A) acid (I) (A) 10AIB Leucine 4-heptanoyl C₃₁H₄₀N₂O₆ 536.7 537 benzyl ester nitrocinnamic chloride (B)(A) acid (I) 10AIC Leucine 4- decanoic acid C₃₄H₄₆N₂O₆ 578.7 579 benzylester nitrocinnamic (C) (A) acid (I) 10AID Leucine 4- methyl C₃₃H₄₂N₂O₈594.7 595 benzyl ester nitrocinnamic suberyl (A) acid (I) chloride (D)10AIE Leucine 4- methyl C₃₅H₄₆N₂O₈ 622.8 623 benzyl ester nitrocinnamicsebacoyl (A) acid (I) chloride (E) 10BIA Phenylalanine 4- glutaricC₃₂H₃₂N₂O₈ 572.6 573 benzyl ester nitrocinnamic anhydride (B) acid (I)(A) 10BIB Phenylalanine 4- heptanoyl C₃₄H₃₈N₂O₆ 570.7 571 benzyl esternitrocinnamic chloride (B) (B) acid (I) 10BIC Phenylalanine 4- decanoicacid C₃₇H₄₄N₂O₆ 612.8 613 benzyl ester nitrocinnamic (C) (B) acid (I)10BID Phenylalanine 4- methyl C₃₆H₄₀N₂O₈ 628.7 629 benzyl esternitrocinnamic suberyl (B) acid (I) chloride (D) 10BIE Phenylalanine 4-methyl C₃₈H₄₄N₂O₈ 656.8 657 benzyl ester nitrocinnamic sebacoyl (B) acid(I) chloride (E) 10AJA Leucine 5-(2- glutaric C₃₁H₃₄N₂O₉ 578.6 579benzyl ester nitrophenyl)- anhydride (A) 2-furoic acid (A) (J) 10AJBLeucine 5-(2- heptanoyl C₃₃H₄₀N₂O₇ 576.7 577 benzyl ester nitrophenyl)-chloride (B) (A) 2-furoic acid (J) 10AJC Leucine 5-(2- decanoic acidC₃₆H₄₆N₂O₇ 618.8 619 benzyl ester nitrophenyl)- (C) (A) 2-furoic acid(J) 10AJD Leucine 5-(2- methyl C₃₅H₄₂N₂O₉ 634.7 635 benzyl esternitrophenyl)- suberyl (A) 2-furoic acid chloride (D) (J) 10AJE Leucine5-(2- methyl C₃₇H₄₆N₂O₉ 662.8 663 benzyl ester nitrophenyl)- sebacoyl(A) 2-furoic acid chloride (E) (J) 10BJA Phenylalanine 5-(2- glutaricC₃₄H₃₂N₂O₉ 612.6 613 benzyl ester nitrophenyl)- anhydride (B) 2-furoicacid (A) (J) 10BJB Phenylalanine 5-(2- heptanoyl C₃₆H₃₈N₂O₇ 610.7 611benzyl ester nitrophenyl)- chloride (B) (B) 2-furoic acid (J) 10BJCPhenylalanine 5-(2- decanoic acid C₃₉H₄₄N₂O₇ 652.8 653 benzyl esternitrophenyl)- (C) (B) 2-furoic acid (J) 10BJD Phenylalanine 5-(2- methylC₃₈H₄₀N₂O₉ 668.7 669 benzyl ester nitrophenyl)- suberyl (B) 2-furoicacid chloride (D) (J) 10BJE Phenylalanine 5-(2- methyl C₄₀H₄₄N₂O₉ 696.8697 benzyl ester nitrophenyl)- sebacoyl (B) 2-furoic acid chloride (E)(J) 10AKA Leucine 5-(3- glutaric C₃₁H₃₄N₂O₉ 578.6 579 benzyl esternitrophenyl)- anhydride (A) 2-furoic acid (A) (K) 10AKB Leucine 5-(3-heptanoyl C₃₃H₄₀N₂O₇ 576.7 577 benzyl ester nitrophenyl)- chloride (B)(A) 2-furoic acid (K) 10AKC Leucine 5-(3- decanoic acid C₃₆H₄₆N₂O₇ 618.8619 benzyl ester nitrophenyl)- (C) (A) 2-furoic acid (K) 10AKD Leucine5-(3- methyl C₃₅H₄₂N₂O₉ 634.7 635 benzyl ester nitrophenyl)- suberyl (A)2-furoic acid chloride (D) (K) 10AKE Leucine 5-(3- methyl C₃₇H₄₆N₂O₉662.8 663 benzyl ester nitrophenyl)- sebacoyl (A) 2-furoic acid chloride(E) (K) 10BKA Phenylalanine 5-(3- glutaric C₃₄H₃₂N₂O₉ 612.6 613 benzylester nitrophenyl)- anhydride (B) 2-furoic acid (A) (K) 10BKBPhenylalanine 5-(3- heptanoyl C₃₆H₃₈N₂O₇ 610.7 611 benzyl esternitrophenyl)- chloride (B) (B) 2-furoic acid (K) 10BKC Phenylalanine5-(3- decanoic acid C₃₉H₄₄N₂O₇ 652.8 653 benzyl ester nitrophenyl)- (C)(B) 2-furoic acid (K) 10BKD Phenylalanine 5-(3- methyl C₃₈H₄₀N₂O₉ 668.7669 benzyl ester nitrophenyl)- suberyl (B) 2-furoic acid chloride (D)(K) 10BKE Phenylalanine 5-(3- methyl C₄₀H₄₄N₂O₉ 696.8 697 benzyl esternitrophenyl)- sebacoyl (B) 2-furoic acid chloride (E) (K) 10ALA Leucine5-(4- glutaric C₃₁H₃₄N₂O₉ 578.6 579 benzyl ester nitrophenyl)- anhydride(A) 2-furoic acid (A) (L) 10ALB Leucine 5-(4- heptanoyl C₃₃H₄₀N₂O₇ 576.7577 benzyl ester nitrophenyl)- chloride (B) (A) 2-furoic acid (L) 10ALCLeucine 5-(4- decanoic acid C₃₆H₄₆N₂O₇ 618.8 619 benzyl esternitrophenyl)- (C) (A) 2-furoic acid (L) 10ALD Leucine 5-(4- methylC₃₅H₄₂N₂O₉ 634.7 635 benzyl ester nitrophenyl)- suberyl (A) 2-furoicacid chloride (D) (L) 10ALE Leucine 5-(4- methyl C₃₇H₄₆N₂O₉ 662.8 663benzyl ester nitrophenyl)- sebacoyl (A) 2-furoic acid chloride (E) (L)10BLA Phenylalanine 5-(4- glutaric C₃₄H₃₂N₂O₉ 612.6 613 benzyl esternitrophenyl)- anhydride (B) 2-furoic acid (A) (L) 10BLB Phenylalanine5-(4- heptanoyl C₃₆H₃₈N₂O₇ 610.7 611 benzyl ester nitrophenyl)- chloride(B) (B) 2-furoic acid (L) 10BLC Phenylalanine 5-(4- decanoic acidC₃₉H₄₄N₂O₇ 652.8 653 benzyl ester nitrophenyl)- (C) (B) 2-furoic acid(L) 10BLD Phenylalanine 5-(4- methyl C₃₈H₄₀N₂O₉ 668.7 669 benzyl esternitrophenyl)- suberyl (B) 2-furoic acid chloride (D) (L) 10BLEPhenylalanine 5-(4- methyl C₄₀H₄₄N₂O₉ 696.8 697 benzyl esternitrophenyl)- sebacoyl (B) 2-furoic acid chloride (E) (L)

Example 6 Synthesis of Representative Compounds of Structure (I)

[0121] Using the same procedures as illustrated in Example 1, additionalrepresentative compounds were prepared using leucine benzyl ester (A) orphenylalanine benzyl ester (B) as the first component, and using varioussecond components to illustrate embodiments wherein the “A” moiety ofstructure (I) is other than a direct bond. The corresponding compoundsare listed below in Table 6. TABLE 6 REPRESENTATIVE COMPOUNDS (R₁ OFSTRUCTURE (I) = OH) MW First Second Third MW found Cpd. No. ComponentComponent Component Formula Calcd (M − H) 11AAA Leucine 2-nitrophenyl-glutaric C₂₁H₂₈N₂O₈ 436.5 437 benzyl ester acetic acid (A) anhydride (A)(A) 11AAB Leucine 2-nitrophenyl- heptanoyl C₂₃H₃₄N₂O₆ 434.5 435 benzylester acetic acid (A) chloride (B) (A) 11AAC Leucine 2-nitrophenyl-decanoic C₂₆H₄₀N₂O₆ 476.6 477 benzyl ester acetic acid (A) acid (C) (A)11AAD Leucine 2-nitrophenyl- methyl C₂₅H₃₆N₂O₈ 492.6 493 benzyl esteracetic acid (A) suberyl (A) chloride (D) 11AAE Leucine 2-nitrophenyl-methyl C₂₇H₄₀N₂O₈ 520.6 521 benzyl ester acetic acid (A) sebacoyl (A)chloride (E) 11BAA Phenylalanine 2-nitrophenyl- glutaric C₂₄H₂₆N₂O₈470.5 471 benzyl ester acetic acid (A) anhydride (B) (A) 11BABPhenylalanine 2-nitrophenyl- heptanoyl C₂₆H₃₂N₂O₆ 468.5 469 benzyl esteracetic acid (A) chloride (B) (B) 11BAC Phenylalanine 2-nitrophenyl-decanoic C₂₉H₃₈N₂O₆ 510.6 511 benzyl ester acetic acid (A) acid (C) (B)11BAD Phenylalanine 2-nitrophenyl- methyl C₂₈H₃₄N₂O₈ 526.6 527 benzylester acetic acid (A) suberyl (B) chloride (D) 11BAE Phenylalanine2-nitrophenyl- methyl C₃₀H₃₈N₂O₈ 554.6 555 benzyl ester acetic acid (A)sebacoyl (B) chloride (E) 11ABA Leucine 3-nitrophenyl- glutaricC₂₁H₂₈N₂O₈ 436.5 437 benzyl ester acetic acid (B) anhydride (A) (A)11ABB Leucine 3-nitrophenyl- heptanoyl C₂₃H₃₄N₂O₆ 434.5 435 benzyl esteracetic acid (B) chloride (B) (A) 11ABC Leucine 3-nitrophenyl- decanoicC₂₆H₄₀N₂O₆ 476.6 477 benzyl ester acetic acid (B) acid (C) (A) 11ABDLeucine 3-nitrophenyl- methyl C₂₅H₃₆N₂O₈ 492.6 493 benzyl ester aceticacid (B) suberyl (A) chloride (D) 11ABE Leucine 3-nitrophenyl- methylC₂₇H₄₀N₂O₈ 520.6 521 benzyl ester acetic acid (B) sebacoyl (A) chloride(E) 11BBA Phenylalanine 3-nitrophenyl- glutaric C₂₄H₂₆N₂O₈ 470.5 471benzyl ester acetic acid (B) anhydride (B) (A) 11BBB Phenylalanine3-nitrophenyl- heptanoyl C₂₆H₃₂N₂O₆ 468.5 469 benzyl ester acetic acid(B) chloride (B) (B) 11BBC Phenylalanine 3-nitrophenyl- decanoicC₂₉H₃₈N₂O₆ 510.6 511 benzyl ester acetic acid (B) acid (C) (B) 11BBDPhenylalanine 3-nitrophenyl- methyl C₂₈H₃₄N₂O₈ 526.6 527 benzyl esteracetic acid (B) suberyl (B) chloride (D) 11BBE Phenylalanine3-nitrophenyl- methyl C₃₀H₃₈N₂O₈ 554.6 555 benzyl ester acetic acid (B)sebacoyl (B) chloride (E) 11ACA Leucine 4-nitrophenyl- glutaricC₂₁H₂₈N₂O₈ 436.5 437 benzyl ester acetic acid (C) anhydride (A) (A)11ACB Leucine 4-nitrophenyl- heptanoyl C₂₃H₃₄N₂O₆ 434.5 435 benzyl esteracetic acid (C) chloride (B) (A) 11ACC Leucine 4-nitrophenyl- decanoicC₂₆H₄₀N₂O₆ 476.6 477 benzyl ester acetic acid (C) acid (C) (A) 11ACDLeucine 4-nitrophenyl- methyl C₂₅H₃₆N₂O₈ 492.6 493 benzyl ester aceticacid (C) suberyl (A) chloride (D) 11ACE Leucine 4-nitrophenyl- methylC₂₇H₄₀N₂O₈ 520.6 521 benzyl ester acetic acid (C) sebacoyl (A) chloride(E) 11BCA Phenylalanine 4-nitrophenyl- glutaric C₂₄H₂₆N₂O₈ 470.5 471benzyl ester acetic acid (C) anhydride (B) (A) 11BCB Phenylalanine4-nitrophenyl- heptanoyl C₂₆H₃₂N₂O₆ 468.5 469 benzyl ester acetic acid(C) chloride (B) (B) 11BCC Phenylalanine 4-nitrophenyl- decanoicC₂₉H₃₈N₂O₆ 510.6 511 benzyl ester acetic acid (C) acid (C) (B) 11BCDPhenylalanine 4-nitrophenyl- methyl C₂₈H₃₄N₂O₈ 526.6 527 benzyl esteracetic acid (C) suberyl (B) chloride (D) 11BCE Phenylalanine4-nitrophenyl- methyl C₃₀H₃₈N₂O₈ 554.6 555 benzyl ester acetic acid (C)sebacoyl (B) chloride (E) 11ADA Leucine 2- glutaric C₂₁H₂₈N₂O₉ 452.5 453benzyl ester nitrophenoxy- anhydride (A) acetic acid (D) (A) 11ADBLeucine 2- heptanoyl C₂₃H₃₄N₂O₇ 450.5 451 benzyl ester nitrophenoxy-chloride (B) (A) acetic acid (D) 11ADC Leucine 2- decanoic C₂₆H₄₀N₂O₇492.6 493 benzyl ester nitrophenoxy- acid (C) (A) acetic acid (D) 11ADDLeucine 2- methyl C₂₅H₃₆N₂O₉ 508.6 509 benzyl ester nitrophenoxy-suberyl (A) acetic acid (D) chloride (D) 11ADE Leucine 2- methylC₂₇H₄₀N₂O₉ 536.6 537 benzyl ester nitrophenoxy- sebacoyl (A) acetic acid(D) chloride (E) 11BDA Phenylalanine 2- glutaric C₂₄H₂₆N₂O₉ 486.5 487benzyl ester nitrophenoxy- anhydride (B) acetic acid (D) (A) 11BDBPhenylalanine 2- heptanoyl C₂₆H₃₂N₂O₇ 484.5 485 benzyl esternitrophenoxy- chloride (B) (B) acetic acid (D) 11BDC Phenylalanine 2-decanoic C₂₉H₃₈N₂O₇ 526.6 527 benzyl ester nitrophenoxy- acid (C) (B)acetic acid (D) 11BDD Phenylalanine 2- methyl C₂₈H₃₄N₂O₉ 542.6 543benzyl ester nitrophenoxy- suberyl (B) acetic acid (D) chloride (D)11BDE Phenylalanine 2- methyl C₃₀H₃₈N₂O₉ 570.6 571 benzyl esternitrophenoxy- sebacoyl (B) acetic acid (D) chloride (E) 11AEA Leucine 3-glutaric C₂₁H₂₈N₂O₉ 452.5 453 benzyl ester nitrophenoxy- anhydride (A)acetic acid (E) (A) 11AEB Leucine 3- heptanoyl C₂₃H₃₄N₂O₇ 450.5 451benzyl ester nitrophenoxy- chloride (B) (A) acetic acid (E) 11AECLeucine 3- decanoic C₂₆H₄₀N₂O₇ 492.6 493 benzyl ester nitrophenoxy- acid(C) (A) acetic acid (E) 11AED Leucine 3- methyl C₂₅H₃₆N₂O₉ 508.6 509benzyl ester nitrophenoxy- suberyl (A) acetic acid (E) chloride (D)11AEE Leucine 3- methyl C₂₇H₄₀N₂O₉ 536.6 537 benzyl ester nitrophenoxy-sebacoyl (A) acetic acid (E) chloride (E) 11BEA Phenylalanine 3-glutaric C₂₄H₂₆N₂O₉ 486.5 487 benzyl ester nitrophenoxy- anhydride (B)acetic acid (E) (A) 11BEB Phenylalanine 3- heptanoyl C₂₆H₃₂N₂O₇ 484.5485 benzyl ester nitrophenoxy- chloride (B) (B) acetic acid (E) 11BECPhenylalanine 3- decanoic C₂₉H₃₈N₂O₇ 526.6 527 benzyl esternitrophenoxy- acid (C) (B) acetic acid (E) 11BED Phenylalanine 3- methylC₂₈H₃₄N₂O₉ 542.6 543 benzyl ester nitrophenoxy- suberyl (B) acetic acid(E) chloride (D) 11BEE Phenylalanine 3- methyl C₃₀H₃₈N₂O₉ 570.6 571benzyl ester nitrophenoxy- sebacoyl (B) acetic acid (E) chloride (E)11AFA Leucine 4- glutaric C₂₁H₂₈N₂O₉ 452.5 453 benzyl esternitrophenoxy- anhydride (A) acetic acid (F) (A) 11AFB Leucine 4-heptanoyl C₂₃H₃₄N₂O₇ 450.5 451 benzyl ester nitrophenoxy- chloride (B)(A) acetic acid (F) 11AFC Leucine 4- decanoic C₂₆H₄₀N₂O₇ 492.6 493benzyl ester nitrophenoxy- acid (C) (A) acetic acid (F) 11AFD Leucine 4-methyl C₂₅H₃₆N₂O₉ 508.6 509 benzyl ester nitrophenoxy- suberyl (A)acetic acid (F) chloride (D) 11AFE Leucine 4- methyl C₂₇H₄₀N₂O₉ 536.6537 benzyl ester nitrophenoxy- sebacoyl (A) acetic acid (F) chloride (E)11BFA Phenylalanine 4- glutaric C₂₄H₂₆N₂O₉ 486.5 487 benzyl esternitrophenoxy- anhydride (B) acetic acid (F) (A) 11BFB Phenylalanine 4-heptanoyl C₂₆H₃₂N₂O₇ 484.5 485 benzyl ester nitrophenoxy- chloride (B)(B) acetic acid (F) 11BFC Phenylalanine 4- decanoic C₂₉H₃₈N₂O₇ 526.6 527benzyl ester nitrophenoxy acid (C) (B) acetic acid (F) 11BFDPhenylalanine 4- methyl C₂₈H₃₄N₂O₉ 542.6 543 benzyl ester nitrophenoxy-suberyl (B) acetic acid (F) chloride (D) 11BFE Phenylalanine 4- methylC₃₀H₃₈N₂O₉ 570.6 571 benzyl ester nitrophenoxy- sebacoyl (B) acetic acid(F) chloride (E) 11AGA Leucine 2- glutaric C₂₂H₂₈N₂O₈ 448.5 449 benzylester nitrocinnamic anhydride (A) acid (G) (A) 11AGB Leucine 2-heptanoyl C₂₄H₃₄N₂O₆ 446.5 447 benzyl ester nitrocinnamic chloride (B)(A) acid (G) 11AGC Leucine 2- decanoic C₂₇H₄₀N₂O₆ 488.6 489 benzyl esternitrocinnamic acid (C) (A) acid (G) 11AGD Leucine 2- methyl C₂₆H₃₆N₂O₈504.6 505 benzyl ester nitrocinnamic suberyl (A) acid (G) chloride (D)11AGE Leucine 2- methyl C₂₈H₄₀N₂O₈ 532.6 533 benzyl ester nitrocinnamicsebacoyl (A) acid (G) chloride (E) 11BGA Phenylalanine 2- glutaricC₂₅H₂₆N₂O₈ 482.5 483 benzyl ester nitrocinnamic anhydride (B) acid (G)(A) 11BGB Phenylalanine 2- heptanoyl C₂₇H₃₂N₂O₆ 480.6 481 benzyl esternitrocinnamic chloride (B) (B) acid (G) 11BGC Phenylalanine 2- decanoicC₃₀H₃₈N₂O₆ 522.6 523 benzyl ester nitrocinnamic acid (C) (B) acid (G)11BGD Phenylalanine 2- methyl C₂₉H₃₄N₂O₈ 538.6 539 benzyl esternitrocinnamic suberyl (B) acid (G) chloride (D) 11BGE Phenylalanine 2-methyl C₃₁H₃₈N₂O₈ 566.6 567 benzyl ester nitrocinnamic sebacoyl (B) acid(G) chloride (E) 11AHA Leucine 3- glutaric C₂₂H₂₈N₂O₈ 448.5 449 benzylester nitrocinnamic anhydride (A) acid (H) (A) 11AHB Leucine 3-heptanoyl C₂₄H₃₄N₂O₆ 446.5 447 benzyl ester nitrocinnamic chloride (B)(A) acid (H) 11AHC Leucine 3- decanoic C₂₇H₄₀N₂O₆ 488.6 489 benzyl esternitrocinnamic acid (C) (A) acid (H) 11AHD Leucine 3- methyl C₂₆H₃₆N₂O₈504.6 505 benzyl ester nitrocinnamic suberyl (A) acid (H) chloride (D)11AHE Leucine 3- methyl C₂₈H₄₀N₂O₈ 532.6 533 benzyl ester nitrocinnamicsebacoyl (A) acid (H) chloride (E) 11BHA Phenylalanine 3- glutaricC₂₅H₂₆N₂O₈ 482.5 483 benzyl ester nitrocinnamic anhydride (B) acid (H)(A) 11BHB Phenylalanine 3- heptanoyl C₂₇H₃₂N₂O₆ 480.6 481 benzyl esternitrocinnamic chloride (B) (B) acid (H) 11BHC Phenylalanine 3- decanoicC₃₀H₃₈N₂O₆ 522.6 523 benzyl ester nitrocinnamic acid (C) (B) acid (H)11BHD Phenylalanine 3- methyl C₂₉H₃₄N₂O₈ 538.6 539 benzyl esternitrocinnamic suberyl (B) acid (H) chloride (D) 11BHE Phenylalanine 3-methyl C₃₁H₃₈N₂O₈ 566.6 567 benzyl ester nitrocinnamic sebacoyl (B) acid(H) chloride (E) 11AIA Leucine 4- glutaric C₂₂H₂₈N₂O₈ 448.5 449 benzylester nitrocinnamic anhydride (A) acid (I) (A) 11AIB Leucine 4-heptanoyl C₂₄H₃₄N₂O₆ 446.5 447 benzyl ester nitrocinnamic chloride (B)(A) acid (I) 11AIC Leucine 4- decanoic C₂₇H₄₀N₂O₆ 488.6 489 benzyl esternitrocinnamic acid (C) (A) acid (I) 11AID Leucine 4- methyl C₂₆H₃₆N₂O₈504.6 505 benzyl ester nitrocinnamic suberyl (A) acid (I) chloride (D)11AIE Leucine 4- methyl C₂₈H₄₀N₂O₈ 532.6 533 benzyl ester nitrocinnamicsebacoyl (A) acid (I) chloride (E) 11BIA Phenylalanine 4- glutaricC₂₅H₂₆N₂O₈ 482.5 483 benzyl ester nitrocinnamic anhydride (B) acid (I)(A) 11BIB Phenylalanine 4- heptanoyl C₂₇H₃₂N₂O₆ 480.6 481 benzyl esternitrocinnamic chloride (B) (B) acid (I) 11BIC Phenylalanine 4- decanoicC₃₀H₃₈N₂O₆ 522.6 523 benzyl ester nitrocinnamic acid (C) (B) acid (I)11BID Phenylalanine 4- methyl C₂₉H₃₄N₂O₈ 538.6 539 benzyl esternitrocinnamic suberyl (B) acid (I) chloride (D) 11BIE Phenylalanine 4-methyl C₃₁H₃₈N₂O₈ 566.6 567 benzyl ester nitrocinnamic sebacoyl (B) acid(I) chloride (E) 11AJA Leucine 5-(2- glutaric C₂₄H₂₈N₂O₉ 488.5 489benzyl ester nitrophenyl)-2- anhydride (A) furoic acid (J) (A) 11AJBLeucine 5-(2- heptanoyl C₂₆H₃₄N₂O₇ 486.6 487 benzyl esternitrophenyl)-2- chloride (B) (A) furoic acid (J) 11AJC Leucine 5-(2-decanoic C₂₉H₄₀N₂O₇ 528.6 529 benzyl ester nitrophenyl)-2- acid (C) (A)furoic acid (J) 11AJD Leucine 5-(2- methyl C₂₈H₃₆N₂O₉ 544.6 545 benzylester nitrophenyl)-2- suberyl (A) furoic acid (J) chloride (D) 11AJELeucine 5-(2- methyl C₃₀H₄₀N₂O₉ 572.7 573 benzyl ester nitrophenyl)-2-sebacoyl (A) furoic acid (J) chloride (E) 11BJA Phenylalanine 5-(2-glutaric C₂₇H₂₆N₂O₉ 522.5 523 benzyl ester nitrophenyl)-2- anhydride (B)furoic acid (J) (A) 11BJB Phenylalanine 5-(2- heptanoyl C₂₉H₃₂N₂O₇ 520.6521 benzyl ester nitrophenyl)-2- chloride (B) (B) furoic acid (J) 11BJCPhenylalanine 5-(2- decanoic C₃₂H₃₈N₂O₇ 562.7 563 benzyl esternitrophenyl)-2- acid (C) (B) furoic acid (J) 11BJD Phenylalanine 5-(2-methyl C₃₁H₃₄N₂O₉ 578.6 579 benzyl ester nitrophenyl)-2- suberyl (B)furoic acid (J) chloride (D) 11BJE Phenylalanine 5-(2- methyl C₃₃H₃₈N₂O₉606.7 607 benzyl ester nitrophenyl)-2- sebacoyl (B) furoic acid (J)chloride (E) 11AKA Leucine 5-(3- glutaric C₂₄H₂₈N₂O₉ 488.5 489 benzylester nitrophenyl)-2- anhydride (A) furoic acid (K) (A) 11AKB Leucine5-(3- heptanoyl C₂₆H₃₄N₂O₇ 486.6 487 benzyl ester nitrophenyl)-2-chloride (B) (A) furoic acid (K) 11AKC Leucine 5-(3- decanoic C₂₉H₄₀N₂O₇528.6 529 benzyl ester nitrophenyl)-2- acid (C) (A) furoic acid (K)11AKD Leucine 5-(3- methyl C₂₈H₃₆N₂O₉ 544.6 545 benzyl esternitrophenyl)-2- suberyl (A) furoic acid (K) chloride (D) 11AKE Leucine5-(3- methyl C₃₀H₄₀N₂O₉ 572.7 573 benzyl ester nitrophenyl)-2- sebacoyl(A) furoic acid (K) chloride (E) 11BKA Phenylalanine 5-(3- glutaricC₂₇H₂₆N₂O₉ 522.5 523 benzyl ester nitrophenyl)-2- anhydride (B) furoicacid (K) (A) 11BKB Phenylalanine 5-(3- heptanoyl C₂₉H₃₂N₂O₇ 520.6 521benzyl ester nitrophenyl)-2- chloride (B) (B) furoic acid (K) 11BKCPhenylalanine 5-(3- decanoic C₃₂H₃₈N₂O₇ 562.7 563 benzyl esternitrophenyl)-2- acid (C) (B) furoic acid (K) 11BKD Phenylalanine 5-(3-methyl C₃₁H₃₄N₂O₉ 578.6 579 benzyl ester nitrophenyl)-2- suberyl (B)furoic acid (K) chloride (D) 11BKE Phenylalanine 5-(3- methyl C₃₃H₃₈N₂O₉606.7 607 benzyl ester nitrophenyl)-2- sebacoyl (B) furoic acid (K)chloride (E) 11ALA Leucine 5-(4- glutaric C₂₄H₂₈N₂O₉ 488.5 489 benzylester nitrophenyl)-2- anhydride (A) furoic acid (L) (A) 11ALB Leucine5-(4- heptanoyl C₂₆H₃₄N₂O₇ 486.6 487 benzyl ester nitrophenyl)-2-chloride (B) (A) furoic acid (L) 11ALC Leucine 5-(4- decanoic C₂₉H₄₀N₂O₇528.6 529 benzyl ester nitrophenyl)-2- acid (C) (A) furoic acid (L)11ALD Leucine 5-(4- methyl C₂₈H₃₆N₂O₉ 544.6 545 benzyl esternitrophenyl)-2- suberyl (A) furoic acid (L) chloride (D) 11ALE Leucine5-(4- methyl C₃₀H₄₀N₂O₉ 572.7 573 benzyl ester nitrophenyl)-2- sebacoyl(A) furoic acid (L) chloride (E) 11BLA Phenylalanine 5-(4- glutaricC₂₇H₂₆N₂O₉ 522.5 523 benzyl ester nitrophenyl)-2- anhydride (B) furoicacid (L) (A) 11BLB Phenylalanine 5-(4- heptanoyl C₂₉H₃₂N₂O₇ 520.6 521benzyl ester nitrophenyl)-2- chloride (B) (B) furoic acid (L) 11BLCPhenylalanine 5-(4- decanoic C₃₂H₃₈N₂O₇ 562.7 563 benzyl esternitrophenyl)-2- acid (C) (B) furoic acid (L) 11BLD Phenylalanine 5-(4-methyl C₃₁H₃₄N₂O₉ 578.6 579 benzyl ester nitrophenyl)-2- suberyl (B)furoic acid (L) chloride (D) 11BLE Phenylalanine 5-(4- methyl C₂₆H₃₂N₂O₉606.7 607 benzyl ester nitrophenyl)-2- sebacoyl (B) furoic acid (L)chloride (E)

Example 7 Biological Activity

[0122] Neuronal Viability Assay

[0123] This assay is used to assess the ability of compounds of thisinvention to protect neurons from glutamate-induced excitotoxic celldeath. Primary cell culture is performed with embryonic day 18 rathippocampal and cortical neurons that are plated into BiocoatPoly-D-Lysine precoated 96-well plates (Becton-Dickinson, Bedford,Mass.; catalog no. 356461) at a density of 21,000 cells/well. The cellsare grown in Neurobasal media (Gibco/Life Technologies, Rockville, Md.,catalog no. 21103049) supplemented with B27, penicillin (100 IU/ml),streptomycin (100 μg/ml) and 500 μM L-glutamine. This media supportsgrowth of pure neuronal cultures as contaminating glial cells that maybe initially present do not survive in the media conditions. At 17 daysafter culture, media is aspirated from wells and 100 μl solution of testcompound in assay buffer (HBSS supplemented with 25 mM HEPES) is added.After 10 min of incubation, 100 μL of test compound solution in assaybuffer supplemented with 200 μM glutamate/20 μM glycine is added. Tenmin later, cells are treated with 100 μl of a 20 μM solution of MK-801(an NMDA receptor antagonist that blocks Ca²⁺ influx; Sigma-Aldrich, St.Louis, Mo., catalog no. M-107) in Neurobasal media. After 24 hrs, theextent of cell death is quantitated by measuring lactate dehydrogenaseactivity released by lysed cells using a calorimetric CytotoxicityDetection Kit (Roche Diagnostics GmbH, Mannheim, Germany, catalog no. 1644 793) and following the manufacturer's instructions.

[0124] In this assay, preferred compounds have a mean neuronal viabilityof 0.6 or less. To this end, preferred compounds of this inventioninclude the following: 6AAD, 6AAE, 6ABE, 6ABF, 6ACE, 6BAA, 6BAE, 6BAG,6BCA, 6BCB, 6CCA, 7AAC, 7AAE, 7ABA, 7BAA, 7BBD, 9AAA, 9AAB, 9AAE, 9ABA,9ABB, 9ABC, 9BAD, 9BAE, 9BBA, 9BBD, 9CAA, 9CAB, 9CAC, 9CAD, 9CBA, 9CBC,9CBD, 9CBE, 9DBA, 9DBB, 9DBC, 8AAB, 8AAE, 8ABB, 8ABE, 8BAB, 8BBA, 8BBB,8CAB, 8CAD and 8CBA.

[0125] Displacement Assays of an Adenine Nucleotide Translocase (ANT)Ligand from Isolated Mitochondria using Test Compounds

[0126] Compound 1 below is a ¹²⁵I-labeled atractyloside derivative thatbinds to the mitochondrial adenine nucleotide translocase with highaffinity (IC₅₀=300 nM in a displacement assay using [³H]-ADP as ligand).Thus, Compound 1 may be used as the radioligand to measure efficacy ofbinding of the compounds of this invention.

[0127] Competition binding assays are performed using bovine cardiacmitochondria. One microgram aliquots of mitochondrial protein areincubated with 100 μl binding buffer (10 mM Tris, 120 mM KCl, 6 mMMgCl₂, 1 mM EDTA, pH 7.4) containing 0.5 nM Compound 1 and a testcompound at various concentrations (10 or 100 μM). Mixtures areincubated for one hour on ice, at the end of which unbound ligand isseparated by centrifugation. Supernatants containing unbound Compound 1are aspirated and discarded. Mitochondrial pellets are washed with fourvolumes of cold binding buffer and counted in a Micromedic 4/200automatic gamma counter. For higher throughput, assays are performed ina 96 well microtitre well format. Unbound ligand is removed byfiltration and washes through glass fiber filter mats (Whatman GF/Bpaper, catalog no. FPXLR-196, Brandel, Inc., Gaithersburg, Md.). Theradioactivity associated with the mitochondrial pellets retained in thefilter mat is determined in a 1450 Wallac MicroBeta TriLux liquidscintillation and luminescence counter (EG&G Wallac, Gaithersburg, Md.).In this assay, preferred compounds of this invention (at 10 μM) displacethe radioligand (i.e., Compound 1) such that 80% or less of theradioactivity of the mitochondrial pellets is detected. To this end,preferred compounds are 6AAB, 6AAD, 6ABA, 6ABB, 6ABD, 6ACB, 6ACC, 6BAB,6BAC, 6BAD, 6BBA, 6BCB, 6BCF, 6CAB, 6CAC, 6CAD, 6CAG, 6CBB, 6CBD, 6CBE,6CCC, 6CCD, 7AAE, 8AAE, 8BBC and 8CBC.

Example 8 Chondrocyte Cytoprotection

[0128] This example illustrates the ability of a test compound, 9DBC, tomediate chondrocyte cytoprotection. More specifically, the test compoundwas found to protect against (1) trigger-induced cell death (i.e.,viability), (2) trigger-induced inhibition of collagen synthesis, (3)trigger-induced GAG release, and (4) IL-1-mediated GAG release and NOgeneration. The procedures employed are set forth below, while theresults of these assays are summarized in Table 7. TABLE 7 SUMMARY OFRESULTS GAG⁴ (Slices) Viability¹ (cells) Collagen² (cells) GAG³ (cells)NO⁴ (Slices) Trigger NOC-12 SIN-1 NOC-12 SIN-1 IL-1 NOC-12 SIN-1 IL-1IL-1 IL-1 ˜EC₅₀ 1 nm 1 nm 1 μm >1 μm >1 μm 100 nm 100 nm 100 nm 10 μm 10μm

[0129] Chondrocyte Function Screening Assays

[0130] Cell culture: Chondrocytic TC28 cells were maintained inmonolayer culture in DMEM/Ham's F12 (1:1) and supplemented with 10% FCS,1% L-glutamine, 100 units/ml Penicillin and 50 mg/ml Streptomycin (OmegaScientific, Tarzana, Calif.) and cultured at 37ƒC with 5% CO₂.Additionally, to further study chondrocytic cells in a more physiologicnonadherent state, in some experiments, TC28 cells were transferred to 6well plates that had been previously coated for 18 hours at 22° C. with10% (v/v) in 95% ethanol solution of the cell adhesion inhibitor poly2-Hydroxyethyl methacrylate (polyHEME), followed by two washes in PBS.Complete DMEM/Ham's F12 medium was then added to the wells and the cellsstudied for up to 72 hours in culture. Type II collagen and aggrecanexpression were confirmed using RT-PCR, which verified maintenance ofchondrocyte phenotype.

[0131] Screening Assays: Compound 9DBC was screened for chondrocyteprotective effects in vitro. The agonists employed have included a donorof nitric oxide (NOC-12, 250 uM), a donor of peroxynitrite (SIN-1, 100uM), and human recombinant IL-1 beta (10 ng/ml). Cytotoxicity wasmeasured via standard lactate dehydrogenase (LDH) release assay asdescribed below. Collagen synthesis was monitored by 3H prolineincorporation into TC28 cells, TCA precipitation of proteins, followedby assay of radioactivity in collagenae sensitive proteibn as outlinedin Johnson et al (Arthritis Rheum. 43:1560-70, 2000). NO was detected byusing the Greiss reaction.

[0132] Cytotoxicity Assay

[0133] 10⁵ TC28 cells (DMEM/F12 media with 10% FCS, 1% glutamine, 1%P/S) were plated each well in a 96 well plate and allowed to adhereovernight. The cells were washed once with PBS and media changed tocontain only 1% FCS. Compound 9DBC at various concentrations was addedto the cells for a pretreatment of 1 hr. The media was removed and freshcompound +/− the toxic stimuli are added. The cells were then incubatedfor 24 hrs at 37° C. Following the incubation the media was collectedand used for analysis in the CytTox 96 Nonradioactive CytotoxicityAssay. Briefly, the LDH release from the dead cells was quantified in a30 min enzymatic reaction that resulted in the conversion of atetrazolium saletin to a red formazan product. The results wereexpressed as the percent of cells dead as to the release of LDH by thecontrol cells.

[0134] Gylcosaminoglycans (GAG) Release Assay

[0135] The enhanced release from chondrocytes of glycosaminoglycans(GAG) is a central feature of osteoarthritic chondrocytes, and is knownto be stimulated potently by IL-1, which, like NO and peroxynitrite isheld to be a major pathogenic factor in osteoarthritis. Thus, GAGrelease assays were carried out on Compound 9DBC, in which, to optimizethe screening assay, a one hour digestion of the cartilage “nodules”formed in the polyheme system was carried out using 300 ug/ml of papainin 20 mM sodium phosphate, 1 mM EDTA, and 2mM DTT (pH 6.8). Thedigestion of the interfering proteins accomplished in this mannerallowed the GAG release to be more readily detectable, and the GAGrelease was quantified by the standard dimethylene blue (DMB) dyebinding colorimetric assay. In brief, the cell extract digested fromabove was combined with 46 uM DMB, 40 mM glycine and 40 mM NaCl (pH 3.0)and immediately read at 525 nm and compared again a standard curve of1-50 ug/ml chondroitin sulfate.

[0136] Bovine Cartilage Organ Culture Methods

[0137] Mature bovine knees were obtained and cartilage from the femoralcondyles and patellar groove was removed in full thickness slices(1-3mm). Circular cores (6-7 mm in diameter) were punched out of thetissue. The cores were washed twice with media (1% FCS, 1% P/S, 1%glutamine containing DMEM high glucose) and then placed in 96 wellsplates. The slices were incubated in media (as above) at 37° C. for 48hrs to allow for recovery from the isolation process. After the recoveryperiod, the media was removed and fresh media with Compound 9DBC wasadded to the slices for a pretreatment period of 6 hrs. Then the mediawas removed and fresh compound plus/minus IL-1 was added and incubatedat 37° C. for 24 hrs. The conditioned media was collected and the GAGand NO release were analyzed. Finally the slices were weighed to correctfor slight variations in size or thickness.

[0138] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1. A compound having the structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof, wherein: A is a direct bond, alkyldiyl, substituted alkyldiyl, —O-(alkyldiyl)-, —O-(substituted alkyldiyl)-, -(alkyldiyl)—O-, -(substituted alkyldiyl)—O-, —N(R′)-(alkyldiyl)-, —N(R′)-(substituted alkyldiyl)-, -(alkyldiyl)-N(R′)-, -(substituted alkyldiyl)- N(R′)-, heterocyclediyl, substituted heterocyclediyl, heterocyclealkyldiyl or substituted heterocyclealkyldiyl, wherein R′ is hydrogen or alkyl; R₁ is hydroxy, alkoxy, aryloxy, arylalkyloxy, amino, or mono- or di-alkylamino; R₂ is hydrogen, alkyl, substituted alky, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl or substituted heterocyclealkyl; and R₃ is alkyl, substituted alky, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl or substituted heterocyclealkyl.
 2. The compound of claim 1 wherein R₁ is hydroxy.
 3. The compound of claim 1 wherein R₁ is alkyoxy.
 4. The compound of claim 3 wherein R₁ is methoxy.
 5. The compound of claim 1 wherein R₁ is amino.
 6. The compound of claim 1 wherein R₁ is aryloxy or arylalkyloxy.
 7. The compound of claim 1 wherein R₂ is hydrogen.
 8. The compound of claim 1 where R₂ is alkyl.
 9. The compound of claim 1 wherein R₃ is alkyl.
 10. The compound of claim 1 wherein R₃ is substituted alkyl.
 11. The compound of claim 1 wherein A is a direct bond.
 12. The compound of claim 1 wherein A is alkyldiyl.
 13. The compound of claim 1 wherein A is —O-(alkyldiyl)- or -(alkyldiyl)—O-.
 14. The compound of claim 1 wherein A is —N(R′)alkyldiyl- or -(alkyldiyl)-N(R′)-.
 15. The compound of claim 1 wherein A is heterocyclediyl.
 16. The compound of claim 1 wherein A is heterocyclealkyldiyl.
 17. A pharmaceutical composition comprising a compound of claim 1 in combination with a pharmaceutically acceptable carrier.
 18. A method for treating a disease by altering mitochondrial function that affects cellular processes, comprising administering to an animal in need thereof an effective amount of the composition of claim
 17. 